High molecular weight major outer membrane protein of Moraxella

ABSTRACT

An isolated and purified outer membrane protein of a Moraxella strain, particularly  M. catarrhalis , having a molecular mass of about 200 kDa, is provided. The about 200 kDa outer membrane protein as well as nucleic acid molecules encoding the same are useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host to confer protection against disease caused by a bacterial pathogen that produces the about 200 kDa outer membrane protein or produces a protein capable of inducing antibodies in a host specifically reactive with the about 200 kDa outer membrane protein.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of copending U.S.patent application Ser. No. 08/478,370, filed Jun. 7, 1995, which itselfis a continuation-in-part of U.S. patent application Ser. No. 08/431,718filed May 1, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of immunology and isparticularly concerned with outer membrane proteins from Moraxella,methods of production thereof, genes encoding such proteins and usesthereof.

BACKGROUND OF THE INVENTION

[0003] Otitis media is the most common illness of early childhood withapproximately 70% of all children suffering at least one bout of otitismedia before the age of seven. Chronic otitis media can lead to hearing,speech and cognitive impairment in children. It is caused by bacterialinfection with Streptococcus pneumoniae (approximately 50%), non-typableHaemophilus influenzae (approximately 30) and Moraxella (Branhamella)catarrhalis (approximately 20%). In the United States alone, treatmentof otitis media costs between one and two billion dollars per year forantibiotics and surgical procedures, such as tonsillectomies,adenoidectomies and insertion of tympanostomy tubes. Because otitismedia occurs at a time in life when language skills are developing at arapid pace, developmental disabilities specifically related to learningand auditory perception have been documented in youngsters with frequentotitis media.

[0004]M. catarrhalis mainly colonizes the respiratory tract and ispredominantly a mucosal pathogen. Studies using cultures of middle earfluid obtained by tympanocentesis have shown that M. catarrhalis causesapproximately 20% of cases of otitis media (ref. 1-Throughout thisapplication, various references are referred to in parenthesis to morefully describe the state of the art to which this invention pertains.Full bibliographic information for each citation is found at the end ofthe specification, immediately preceding the claims. The disclosures ofthese references are hereby incorporated by reference into the presentdisclosure).

[0005] The incidence of otitis media caused by M. catarrhalis isincreasing. As ways of preventing otitis media caused by pneumococcusand non-typable H. influenzae are developed, the relative importance ofM. catarrhalis as a cause of otitis media can be expected to furtherincrease.

[0006]M. catarrhalis is also an important cause of lower respiratorytract infections in adults, particularly in the setting of chronicbronchitis and emphysema (refs. 2, 3, 4, 5, 6, 7, and 8). M. catarrhalisalso causes sinusitis in children and adults (refs. 9, 10. 11, 12, and13) and occasionally causes invasive disease (refs. 14, 15, 16, 17, 18,and 19).

[0007] Like other Gram-negative bacteria, the outer membrane of M.catarrhalis consists of phospholipids, lipopolysaccharide (LPS), andouter membrane proteins (OMPs). Eight of the M. catarrhalis OMPs havebeen identified as major components. These are designated by letters Ato H, beginning with OMP A which has a molecular mass of 98 kDa to OMP Hwhich has a molecular mass of 21 kDa (ref. 20).

[0008] Recently, a high-molecular-weight outer membrane protein of M.catarrhalis was purified and characterized (ref. 21). The apparentmolecular mass of this protein varies from 350 kDa to 720 kDa as judgedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).This protein appears to be an oligomer of much smaller proteins orsubunits thereof of molecular mass 120 to 140 kDa and is antigenicallyconserved among strains of Moraxella.

[0009] A protein molecular mass of about 300 to 400 kDa named UspA wasalso reported to be present on the surface of Moraxella (ref. 22).

[0010]M. catarrhalis infection may lead to serious disease. It would beadvantageous to provide other outer membrane proteins for M. catarrhalisand genes encoding such proteins for use as antigens in immunogenicpreparations including vaccines, carriers for other antigens andimmunogens and the generation of diagnostic reagents.

SUMMARY OF THE INVENTION

[0011] The present invention is directed towards the provision of apurified and isolated major outer membrane protein of Moraxellacatarrhalis and other Moraxella strains, having an apparent molecularmass of about 200 kDa, as well as genes encoding the same.

[0012] In accordance with one aspect of the invention, there is providedan isolated and purified, outer membrane protein of a Moraxella strainhaving a molecular weight of about 200 kDa, as determined by SDS-PAGE,or a fragment or an analog thereof. The outer membrane protein may besubstantially in its native conformation (so as to have substantiallyretained the characteristic immunogenicity of the outer membrane proteinin the Moraxella strain) and may be isolated from a M. catarrhalisstrain, such as from M. catarrhalis 4223. Such isolated and purifiedabout 200 kDa outer membrane protein is substantially free from non-200kDa outer membrane proteins, phospholipids and lipopolysaccharide ofMoraxella. The about 200 kDa outer membrane protein is at least about 70wt % pure, preferably at least about 90 wt % pure, and may be in theform of an aqueous solution thereof. Such about 200 kDa outer membraneprotein may have substantially the amino acid composition shown in TableIII and a deduced amino acid sequence as shown in FIG. 6 (SEQ ID No: 3).

[0013] The present invention also provides a purified and isolatednucleic acid molecule encoding an outer membrane protein of a strain ofMoraxella having a molecular mass of about 200 kDa, as determined bySDS-PAGE, or a fragment or an analog of the outer membrane protein. Theprotein encoded by the nucleic acid molecule may comprise a proteincontaining the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile(SEQ ID No: 5) particularly where X is Lys (SEQ ID No: 10), forMoraxella catarrhalis strain 4223 or containing the corresponding aminoacid sequence from other Moraxella strains.

[0014] In a further aspect of the present invention, there is provided apurified and isolated nucleic acid molecule having a sequence selectedfrom the group consisting of (a) a DNA sequence as set out in FIG. 6(SEQ ID Nos: 1 or 2), or the complementary sequence thereto; (b) a DNAsequence encoding an about 200 kDa protein of a strain of Moraxella andcontaining the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile(SEQ ID No: 5), particularly where x is Lys (SEQ ID No: 10) or thecomplementary sequence thereto; (c) a DNA sequence encoding the deducedamino acid sequence as set out in FIG. 6 (SEQ ID No: 3) or thecomplementary sequence thereto; and (d) a nucleotide sequence whichhybridizes under stringent conditions to any one of the sequencesdefined in (a), (b) or (c). The nucleic acid preferably defined in (d)has at least about 90% sequence identity with any one of the sequencesdefined in (a), (b) or (c).

[0015] The nucleic acid molecules provided herein may be included in avector adapted for transformation of a host. The nucleic acid moleculesprovided herein also may be included in an expression vector adapted fortransformation of a host along with expression means operatively coupledto the nucleic acid molecule for expression by the host of the about 200kDa outer membrane protein of a strain of Moraxella or the fragment orthe analog of the outer membrane protein. A transformed host containingthe expression vector is included within the invention, along with arecombinant outer membrane protein or fragment or analog thereofproducible by the transformed host.

[0016] The expression means may include a nucleic acid portion encodinga leader sequence for secretion from the host of the outer membraneprotein or the fragment or the analog of the outer membrane protein. Theexpression means may include a nucleic acid portion encoding alipidation signal for expression from the host of a lipidated form ofthe outer membrane protein or the fragment or analog thereof.

[0017] The present invention further includes a live vector for deliveryof the outer membrane protein of the invention or a fragment or analogthereof, comprising a vector containing the nucleic acid moleculeprovided herein. The live vector may be selected from the groupconsisting of E. coli, Salmonella, BCG, adenovirus, poxvirus, vacciniaand poliovirus.

[0018] In accordance with a further aspect of the present invention,there is provided a peptide having no less than six amino acids and nomore than 150 amino acids and containing an amino acid sequencecorresponding to a portion only of the outer membrane protein of theinvention, or a fragment or analog thereof. The peptide may be onehaving the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10) for the Moraxella catarrhalis 4223 strain or the aminoacid sequence for the corresponding peptide for other strains ofMoraxella.

[0019] The present invention also provides an immunogenic compositioncomprising an immunoeffective amount of an active component, which maybe the outer membrane protein or fragment or analog thereof, nucleicacid molecules, recombinant outer membrane proteins, fragments oranalogs thereof, live vectors, and/or peptides, as provided herein,along with a pharmaceutically acceptable carrier therefor with theactive component producing an immune response when administered to ahost, which may be a primate, particularly a human.

[0020] The immunogenic composition may be formulated as a vaccine for invivo administration to a host to confer protection against diseasescaused by a bacterial pathogen that produces the about 200 kDa outermembrane protein or produces a protein capable of inducing antibodies inthe host specifically reactive with the about 200 kDa outer membraneprotein. In particular, the bacterial pathogen is a strain of Moraxella,particularly M. catarrhalis.

[0021] The immunogenic composition may be formulated as a microparticlecapsule, ISCOM or liposome preparation. The immunogenic composition maybe used in combination with a targeting molecule for delivery tospecific cells of the immune system as to mucosal surfaces. Sometargeting molecules include vitamin B12 and fragments of bacterialtoxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.) andmonoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barberet al). The immunogenic compositions of the invention (includingvaccines) may further comprise at least one other immunogenic orimmunostimulating material and the immunostimulating material may be atleast one adjuvant.

[0022] Suitable adjuvants for use in the present invention include, (butare not limited to) aluminum phosphate, aluminum hydroxide, QS21, QuilA, derivatives and components thereof, ISCOM matrix, calcium phosphate,calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecylester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP,DC-chol, DDBA and a lipoprotein. Advantageous combinations of adjuvantsare described in copending U.S. patent application Ser. No. 08/261,194filed Jun. 16, 1994 and 08/483,856, filed Jun. 7, 1995, assigned to theassignee hereof and the disclosures of which is incorporated herein byreference thereto. The invention further includes an antibody specificfor the outer membrane protein provided herein producible by immunizinga host with an immunogenic composition as provided herein.

[0023] In a further aspect of the invention, there is provided a methodof generating an immune response in a host comprising administeringthereto an immuno-effective amount of the immunogenic composition asprovided herein. The immune response may be a humoral or a cell-mediatedimmune response. The immune response may provide protection to the hostagainst diseases caused by a bacterial pathogen that produces the about200 kDa outer membrane protein or produces a protein capable of inducingantibodies in the host specifically reactive with the about 200 kDaouter membrane protein. In particular, the pathogen is a strain ofMoraxella, including M. catarrhalis. Hosts in which protection againstdisease may be conferred include primates, including humans.

[0024] The present invention provides, in an additional aspect thereof,a method of producing a vaccine comprising administering the immunogeniccomposition provided herein to a test host to determine an amount and afrequency of administration of the active component to confer protectionagainst disease caused by a bacterial pathogen that produces the about200 kDa outer membrane protein or produces a protein capable of inducingantibodies in the host specifically reactive with the about 200 kDaouter membrane protein, and formulating the active component in a formand amount suitable for administration to a treated host in accordancewith said determined amount and frequency of administration. Inparticular, the pathogen is a strain of Moraxella, including M.catarrhalis. The treated host may be a human.

[0025] A further aspect of the present invention provides a method ofdetermining the presence of nucleic acid encoding an outer membraneprotein of a strain of Moraxella having a molecular mass of about 200kDa, as determined by SDS-PAGE, or fragment or analog thereof, in asample, comprising the steps of:

[0026] (a) contacting the sample with the nucleic acid molecule providedherein to produce duplexes comprising the nucleic acid molecule and anysaid nucleic acid molecule encoding the outer membrane protein presentin the sample and specifically hybridizable therewith; and

[0027] (b) determining the production of the duplexes.

[0028] In yet a further aspect of the invention, there is provided amethod of determining the presence of antibodies specifically reactivewith outer membrane protein of a strain of Moraxella having a molecularmass of about 200 kDa, in a sample, comprising the steps of:

[0029] (a) contacting the sample with the outer membrane protein asprovided herein to produce complexes comprising the outer membraneprotein and any said antibodies present in the sample specificallyreactive therewith; and

[0030] (b) determining production of the complexes.

[0031] In a further aspect of the invention, there is also provided amethod of determining the presence of an outer membrane protein of astrain of Moraxella having a molecular mass of about 200 kDa, in asample comprising the steps of:

[0032] (a) immunizing a subject with the immunogenic composition asprovided herein, to produce antibodies specific for the outer membraneprotein;

[0033] (b) contacting the sample with the antibodies to producecomplexes comprising any outer membrane protein present in the sampleand said outer membrane protein specific antibodies; and

[0034] (c) determining production of the complexes.

[0035] The outer membrane protein may be part of a Moraxella catarrhalisstrain.

[0036] The present invention provides, in a yet further aspect, adiagnostic kit for determining the presence of nucleic acid encoding anouter membrane protein of a strain of Moraxella having a molecular massof about 200 kDa, as determined by SDS-PAGE, or fragment or analogthereof, in a sample, comprising:

[0037] (a) the nucleic acid molecule as provided herein;

[0038] (b) means for contacting the nucleic acid with the sample toproduce duplexes comprising the nucleic acid molecule and any saidnucleic acid present in the sample and hybridizable with the nucleicacid molecule; and

[0039] (c) means for determining production of the duplexes.

[0040] In yet a further aspect of the invention, there is provided adiagnostic kit for determining the presence of antibodies in a samplespecifically reactive with the outer membrane protein of a strain ofMoraxella having a molecular mass of about 200 kDa, as determined bySDS-PAGE, comprising:

[0041] (a) the outer membrane protein as provided herein;

[0042] (b) means for contacting the outer membrane protein with thesample to produce complexes comprising the outer membrane protein andany said antibodies present in the sample; and

[0043] (c) means for determining production of the complexes.

[0044] The invention also provides a diagnostic kit for detecting thepresence of an outer membrane protein of a strain of Moraxella having amolecular mass of about 200 kDa, in a sample, comprising:

[0045] (a) an antibody specific for the about 200 kDa outer membraneprotein as provided herein;

[0046] (b) means for contacting the antibody with the sample to producea complex comprising the outer membrane protein and outermembrane-specific antibody; and

[0047] (c) means for determining production of the complex.

[0048] In a further aspect of the invention, there is provided a methodof producing an isolated and purified outer membrane protein of a strainof Moraxella having a molecular mass of about 200 kDa, as determined bySDS-PAGE, comprising the steps of:

[0049] (a) providing a cell mass of the Moraxella strain;

[0050] (b) disrupting the cell mass to provide a cell lysate;

[0051] (c) fractionating the cell lysate to provide a fractioncontaining the outer membrane protein substantially free from other celllysate components, and

[0052] (d) recovering said outer membrane protein.

[0053] The bacterial strain may be M. catarrhalis. The cell lysate maybe fractionated by gel electrophoresis.

[0054] In this application, the term “about 200 kDa protein” is used todefine a family of outer membrane proteins of Moraxella having amolecular mass of between about 160 and about 230 kDa and includesproteins having variations in their amino acid sequences including thosenaturally occurring in various strains of Moraxella. The purified andisolated DNA molecules comprising a gene encoding the about 200 kDaprotein of the present invention also include those encoding functionalanalogs of the about 200 kDa protein. In this application, a firstprotein is a “functional analog” of a second protein if the firstprotein is immunologically related to and/or has the same function asthe second protein. The functional analog may be, for example, afragment of the protein or a substitution, addition, deletion mutantthereof or a fusion with a second protein.

[0055] Advantages of the present invention include:

[0056] a method for isolating purified about 200 kDa outer membraneprotein of a Moraxella strain that produces the outer membrane protein,including M. catarrhalis;

[0057] a gene encoding an about 200 kDa outer membrane protein of M.catarrhalis;

[0058] an isolated and purified about 200 kDa outer membrane proteinisolatable from a Moraxella strain; and

[0059] diagnostic kits and immunological reagents for specificidentification of Moraxella and hosts infected thereby.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060]FIGS. 1A and 1B show an analysis of Moraxella catarrhalis cellproteins by SDS-PAGE. The identification of the lanes and the sources ofthe proteins are given in Example 2 below;

[0061]FIG. 2 shows a comparative analysis of cell proteins from a numberof M. catarrhalis strains by SDS-PAGE analysis and shows the variabilityin the molecular weight of the about 200 kDa protein in differentstrains of Moraxella. The identification of the lanes and the sources ofthe proteins are given in Example 4 below;

[0062]FIG. 3 shows an analysis of isolated and purified about 200 kDaouter membrane protein of M. catarrhalis by SDS-PAGE;

[0063]FIG. 4 shows the specific recognition of about 200 kDa outermembrane protein by anti-peptide antiserum. The identification of thelanes and antiserum are given in Example 8 below;

[0064]FIG. 5 shows restriction maps of clones containing a gene encodingthe about 200 kDa outer membrane protein of M. catarrhalis. The openreading frame of the about 200 kDa outer membrane protein is indicatedby the shaded box. Restriction sites are Sal: SalI, N: NcoI, B: BglII,K: KpnI, Xh: XhoI, RV: EcoRV.

[0065]FIG. 6 shows the nucleotide sequence (SEQ ID No: 1—entiresequence, SEQ ID No: 2—coding sequence) of the gene encoding the about200 kDa outer membrane protein of M. catarrhalis and the deduced aminoacid sequence (SEQ ID No: 3—identified GTG start codon, SEQ ID No:4—putative ATG start codon). Peptide 1 (SEQ ID No: 11) and Peptide 2(SEQ ID No: 12) are identified by underlining;

[0066]FIG. 7A is a restriction enzyme map of the gene encoding the about200 kDa outer membrane protein of M. catarrhalis (SEQ ID No: 1) showingsingle cutting restriction enzymes;

[0067]FIG. 7B is a restriction enzyme map of the gene encoding about 200kDa outer membrane protein of M. catarrhalis (SEQ ID No: 1) showingdouble cutting restriction enzymes;

[0068]FIG. 8 shows the identification of the GTG initiation codon byexpressing the C-terminal truncations of the gene encoding the about 200kDa outer membrane protein of M. catarrhalis. Restriction sites are N:NcoI, K: KpnI, H: HindIII, Hp: HpaI, RV: EcoRV, Sal: SalI;

[0069]FIG. 9 shows the identification of the GTG initiation codon byutilization of anti-sera specific for N-terminal peptides of the about200 kDa outer membrane protein of M. catarrhalis. Restriction sites areNco: NcoI, K: KpnI, H: HindIII, RV: EcoRV, Sal: SalI;

[0070]FIG. 10 shows the recognition of 200 kDa protein by anti peptidesera;

[0071]FIG. 11 shows the construction of vectors for the expression ofthe about 200 kDa outer membrane protein of M. catarrhalis from E. coli.Nco: NcoI, Pst: PstI, Pvu:PvuII, Sca: ScaI, Sal: SalI;

[0072]FIG. 12 shows the expression of N-terminal truncations of theabout 200 kDa outer membrane protein of M. catarrhalis in E. coli usingthe bacteriophage T7 promoter;

[0073]FIG. 13 shows the expression of the about 200 kDa outer membraneprotein of M. catarrhalis fused with the LacZ-α-peptide in E. coli; and

[0074]FIG. 14 shows the specific identification of M. catarrhalisexpressing the about 200 kDa outer membrane protein by guinea piganti-200 kDa specific antiserum in contrast to other bacteria.Identification of the lanes and bacteria appears below.

GENERAL DESCRIPTION OF THE INVENTION

[0075] Referring to FIG. 1A and 1B and FIG. 2, there is illustrated theseparation of a novel outer membrane protein from a variety of strainsof M. catarrhalis having a molecular mass about 200 kDa. The presence ofthis about 200 kDa protein in a variety of M. catarrhalis strains and,in particular, the almost-universal presence in strains isolated frompatients suffering from otitis media is shown in Table I. FIG. 3 showsthe isolated and purified outer membrane protein.

[0076] Purified protein was eluted from a gel and used to raiseantibodies in guinea pigs. The antibodies specifically recognize onlystrains of M. catarrhalis which produce the outer membrane protein(Table I below).

[0077] Referring to FIG. 4, there is shown the recognition of the about200 kDa outer membrane protein by antibodies raised in guinea pigs to asynthesized peptide corresponding to an internal fragment of the about200 kDa protein. The synthesized peptide had the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQID No: 6).

[0078] Referring to FIG. 5, there is shown restriction maps of clonescontaining a gene encoding the about 200 kDa outer membrane protein. InFIG. 5, the open reading frame of the about 200 kDa gene is shown as asolid box and the GTG start codon is indicated. The nucleotide sequence(SEQ ID No: 1 and 2) of the gene encoding the about 200 kDa outermembrane protein is shown in FIG. 6, along with the deduced amino acidsequence (SEQ ID No: 3) of the protein. Restriction enzyme maps of thegene encoding the about 200 kDa protein are shown in FIGS. 7(A) and7(B). The amino acid composition of the about 200 kDa protein is shownin Table III.

[0079] In one embodiment of the present invention, the isolated andpurified about 200 kDa outer membrane protein as provided herein isuseful for generating antibodies that can be used to specificallydistinguish M. catarrhalis from other bacterial pathogens that causeotitis media and other diseases. Thus referring to FIG. 14, there isillustrated an immunoblot showing the specific reactivity of a guineapig monospecific anti-200 kDa outer membrane protein antiserum producedby immunizing mice with the purified about 200 kDa outer membraneprotein as provided herein. The bacterial lysates analyzed were asfollows: Lane Bacterium Source 1. Molecular Weight Standard 2. M.catarrhalis 4223 middle ear fluid 3. M. catarrhalis RH408 non-clumpingvariant of strain 4223 4. H. influenzae, MinnA strain meningitis isolate5. non-typable H. influenzae, SB12 strain otitis media isolate 6.non-typable H. influenzae, SB33 strain otitis media isolate 7. S.pneumoniae type 6 ATCC 6306 8. S. pneumoniae type 14 ATCC 6314 9. P.aeruginosa 10. E. coli DH5α

[0080] The results shown in FIG. 14 clearly show the usefulness of outermembrane-specific antisera as provided herein to distinguish betweenbacterial pathogens that produce diseases with similar clinicalsymptoms.

[0081] In accordance with another aspect of the present invention, thereis provided a vaccine against Moraxella, comprising animmunogenically-effective amount of the outer membrane protein asprovided herein and a physiologically-acceptable carrier therefor. Theouter membrane protein provided herein also may be used as a carrierprotein for hapten, polysaccharides or peptides to make a conjugatevaccine against antigenic determinants unrelated to the about 200 kDaouter membrane protein.

[0082] The about 200 kDa outer membrane protein provided herein isuseful as a diagnostic reagent, as an antigen for the generation ofanti-outer membrane protein antibodies, or as an antigen for vaccinationagainst the diseases caused by species of Moraxella or for detectinginfection by Moraxella.

[0083] In additional embodiments of the present invention, the about 200kDa outer membrane protein as provided herein may be used as a carriermolecule to prepare chimeric molecules and conjugate vaccines (includingglycoconjugates) against pathogenic bacteria, including encapsulatedbacteria. Thus, for example, glycoconjugates of the present inventionmay be used to confer protection against disease and infection caused byany bacteria having polysaccharide antigens includinglipooligosaccharides (LOS) and polyribosylphosphate (PRP). Suchbacterial pathogens may include, for example, Haemophilus influenzae,Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis,Salmonella typhi, Streptococcus mutants, Cryptococcus neoformans,Klebsiella, Staphylococcus aureus and Pseudomonas aeruginosa. Particularantigens which can be conjugated to outer membrane protein and methodsto achieve such conjugations are described in published PCT applicationWO 94/12641, assigned to the assignee hereof and the disclosure of whichis hereby incorporated by reference thereto.

[0084] In another embodiment, the carrier function of the outer membraneprotein may be used, for example, to induce immunity toward abnormalpolysaccharides of tumor cells, or to produce anti-tumor antibodies thatcan be conjugated to chemotherapeutic or bioactive agents.

[0085] The present invention extends to the use of the nucleic acidmolecules and proteins provided herein as a medicament and in themanufacture of a medicament for the treatment of Moraxella infections.

[0086] In a particular embodiment of the invention, there is provided arecombinant about 200 kDa outer membrane protein of Moraxella orfragment or analog thereof or a fusion protein producible by atransformed host containing at least a portion of the gene encoding theabout 200 kDa protein. Referring to FIG. 11, there is shown recombinantvectors for the production of such proteins. In FIG. 11, the filledboxes show 1.9 kb and 4.8 kb C-terminal regions of 200 kD protein gene,that were inserted into a vector, pT7-7, under the control of thebacteriophage T7 promoter. The small open boxes show seven N-terminalamino acids from the vector in the same reading frame. The shaded boxshows 5.5 kb C-terminal region of 200 kD protein, which contained ATGcodon very close to the N-terminus. This gene fragment was fused to lacZα peptide gene (shown in filled box) under the control of lacZ promoter.The full-length gene, that starts from GTG, is shown in a hatched box.

[0087] Referring to FIG. 12, there is shown the expression of N-terminaltruncations of the about 200 kDa protein in E. coli. E. coli strain,BL21(DE3)/pLysS, carrying plasmid, pKS94, was grown in LB brothcontaining 100 μg/ml ampicillin to the early log phase and then IPTG wasadded. After culturing for 2 more hours, the bacteria were harvested andlysed. The lysates were assayed on Western blot using anti-200 kDprotein guinea pig serum as a first antibody. Other procedures were asin FIG. 5. Lane 1:, prestained molecular weight marker, Lane,2:BL21(DE3)/pLysS carrying pT7-7 with an incorrect insert. Lane 3:L21(DE3)/pLysS carrying pKS94.

[0088] Referring to FIG. 13, there is shown the expression of fusionprotein comprising the β-galactosidase α peptide and a portion of theabout 200 kDa protein in E. coli. E. coli strain, DH5α, carried pKS140.The plasmid pKS140 carried the C-terminal 5.5 kb fragment of 200 kDprotein gene after a N-terminal portion of LacZ-α-peptide in the samereading frame. The E. coli strain was grown to the stationary phase,harvested and then lysed. The lysate was assayed by Western blotting.Lane 1: prestained molecular weight marker, Lane 2: DH5α carrying pKS140(total protein, 0.5 μg), Lane 3: sonicate of M. catarrhalis, strain 4223(total protein, 10 μg).

[0089] It is clearly apparent to one skilled in the art, that thevarious embodiments of the present invention have many applications inthe fields of vaccination, diagnosis, treatment of Moraxella infections,and in the generation of immunological reagents. A further non-limitingdiscussion of such uses is further presented below.

[0090] 1. Vaccine Preparation and Use

[0091] Immunogenic compositions, including those suitable to be used asvaccines, may be prepared from the about 200 kDa outer membrane proteinas disclosed herein, as well as immunological fragments and fusionsthereof, which may be purified from the bacteria or which may beproduced recombinantly. The vaccine elicits an immune response in asubject which produces antibodies, including anti-200 kDa outer membraneprotein antibodies and antibodies that are opsonizing or bactericidal.Should the vaccinated subject be challenged by Moraxella or otherbacteria that produce proteins capable of producing antibodies thatspecifically recognize 200 kDa outer membrane protein, the antibodiesbind to and inactivate the bacterium. Furthermore, opsonizing orbactericidal anti-200 kDa outer membrane protein antibodies may alsoprovide protection by alternative mechanisms.

[0092] Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The about 200 kDa outermembrane protein may be mixed with pharmaceutically acceptableexcipients which are compatible with the about 200 kDa outer membraneprotein. Such excipients may include, water, saline, dextrose, glycerol,ethanol, and combinations thereof. The immunogenic compositions andvaccines may further contain auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, or adjuvants to enhance theeffectiveness thereof. Immunogenic compositions and vaccines may beadministered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Oral formulationsmay include normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the about 200 kDa outer membrane protein. Theimmunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, and if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art and may be of the orderof micrograms of the about 200 kDa outer membrane protein. Suitableregimes for initial administration and booster doses are also variable,but may include an initial administration followed by subsequentadministrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host.

[0093] The immunogenic preparations including vaccines may comprise asthe immunostimulating material a nucleotide vector comprising at least aportion of the gene encoding the about 200 kDa protein, or the at leasta portion of the gene may be used directly for immunization.

[0094] The concentration of the about 200 kDa outer membrane antigen inan immunogenic composition according to the invention is in generalabout 1 to 95%. A vaccine which contains antigenic material of only onepathogen is a monovalent vaccine. Vaccines which contain antigenicmaterial of several pathogens are combined vaccines and also belong tothe present invention. Such combined vaccines contain, for example,material from various pathogens or from various strains of the samepathogen, or from combinations of various pathogens.

[0095] Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

[0096] Immunostimulatory agents or adjuvants have been used for manyyears to improve the host immune responses to, for example, vaccines.Intrinsic adjuvants, such as lipopolysaccharides, normally are thecomponents of the killed or attenuated bacteria used as vaccines.Extrinsic adjuvants are immunomodulators which are typicallynon-covalently linked to antigens and are formulated to enhance the hostimmune responses. Thus, adjuvants have been identified that enhance theimmune response to antigens delivered parenterally. Some of theseadjuvants are toxic, however, and can cause undesirable side-effects,making them unsuitable for use in humans and many animals. Indeed, onlyaluminum hydroxide and aluminum phosphate (collectively commonlyreferred to as alum) are routinely used as adjuvants in human andveterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established and aHBsAg vaccine has been adjuvanted with alum. While the usefulness ofalum is well established for some applications, it has limitations. Forexample, alum is ineffective for influenza vaccination andinconsistently elicits a cell mediated immune response.

[0097] A wide range of extrinsic adjuvants can provoke potent immuneresponses to antigens. These include saponins complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria in mineral oil, Freund's completeadjuvant, bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

[0098] To efficiently induce humoral immune responses (HIR) andcell-mediated immunity (CMI), immunogens are typically emulsified inadjuvants. Many adjuvants are toxic, inducing granulomas, acute andchronic inflammations (Freund's complete adjuvant) FCA, cytolysis(saponins and Pluronic polymers) and pyrogenicity, arthritis andanterior uveitis (LPS and MDP). Although FCA is an excellent adjuvantand widely used in research, it is not licensed for use in human orveterinary vaccines because of its toxicity.

[0099] Desirable characteristics of ideal adjuvants include:

[0100] (1) lack of toxicity;

[0101] (2) ability to stimulate a long-lasting immune response;

[0102] (3) simplicity of manufacture and stability in long-term storage;

[0103] (4) ability to elicit both CMI and HIR to antigens administeredby various routes, if required;

[0104] (5) synergy with other adjuvants;

[0105] (6) capability of selectively interacting with populations ofantigen presenting cells (APC);

[0106] (7) ability to specifically elicit appropriate T_(H)1 or T_(H)2cell-specific immune responses; and

[0107] (8) ability to selectively increase appropriate antibody isotypelevels (for example, IgA) against antigens.

[0108] U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989which is incorporated herein by reference thereto, teaches glycolipidanalogues including N-glycosylamides, N-glycosylureas andN-glycosylcarbamates, each of which is substituted in the sugar residueby an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff etal. (U.S. Pat. No. 4,855,283 and ref. 27) reported that N-glycolipidanalogs displaying structural similarities to the naturally-occurringglycolipids, such as glycosphospholipids and glycoglycerolipids, arecapable of eliciting strong immune responses in both herpes simplexvirus vaccine and pseudorabies virus vaccine. Some glycolipids have beensynthesized from long chain-alkylamines and fatty acids that are linkeddirectly with the sugars through the anomeric carbon atom, to mimic thefunctions of the naturally occurring lipid residues.

[0109] U.S. Pat. No. 4,258,029 granted to Moloney, assigned to theassignee hereof and incorporated herein by reference thereto, teachesthat octadecyl tyrosine hydrochloride (OTH) functioned as an adjuvantwhen complexed with tetanus toxoid and formalin inactivated type I, IIand III poliomyelitis virus vaccine. Also, Nixon-George et al. (ref.24), reported that octadecyl esters of aromatic amino acids complexedwith a recombinant hepatitis B surface antigen, enhanced the host immuneresponses against hepatitis B virus.

[0110] Lipidation of synthetic peptides has also been used to increasetheir immunogenicity. Thus, Wiesmuller (ref. 25) describes a peptidewith a sequence homologous to a foot-and-mouth disease viral proteincoupled to an adjuvant tripalmityl-S-glyceryl-cysteinylserylserine,being a synthetic analogue of the N-terminal part of the lipoproteinfrom Gram negative bacteria. Furthermore, Deres et al. (ref. 26)reported in vivo priming of virus-specific cytotoxic T lymphocytes withsynthetic lipopeptide vaccine which comprised of modified syntheticpeptides derived from influenza virus nucleoprotein by linkage to alipopeptide, N-palmityl-S-[2,3-bis(palmitylxy)-(2RS)-propyl-[R]-cysteine(TPC).

[0111] 2. Immunoassays

[0112] The about 200 kDa outer membrane protein of the present inventionis useful as an immunogen for the generation of anti-200 kDa outermembrane protein antibodies, as an antigen in immunoassays includingenzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzymelinked antibody binding assays or procedures known in the art for thedetection of anti-bacterial, anti-Moraxella, and anti-200 kDa outermembrane protein antibodies. In ELISA assays, the about 200 kDa outermembrane protein is immobilized onto a selected surface, for example, asurface capable of binding proteins such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbed about200 kDa outer membrane protein, a nonspecific protein such as a solutionof bovine serum albumin (BSA) that is known to be antigenically neutralwith regard to the test sample may be bound to the selected surface.This allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific bindings of antisera onto the surface.

[0113] The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from 2 to 4 hours, at temperatures suchas of the order of about 20° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound about 200 kDaouter membrane protein, and subsequent washing, the occurrence, and evenamount, of immunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity such as an enzymatic activity that will generate,for example, a colour development upon incubating with an appropriatechromogenic substrate. Quantification may then be achieved by measuringthe degree of colour generation using, for example, a visiblespectrophotometer.

[0114] 3. Use of Sequences as Hybridization Probes

[0115] The nucleotide sequences of the present invention, comprising thesequence of the about 200 kDa protein gene, now allow for theidentification and cloning of the about 200 kDa protein gene from anyspecies of Moraxella.

[0116] The nucleotide sequences comprising the sequence of the about 200kDa protein gene of the present invention are useful for their abilityto selectively form duplex molecules with complementary stretches ofother about 200 kDa protein genes. Depending on the application, avariety of hybridization conditions may be employed to achieve varyingdegrees of selectivity of the probe toward the other genes. For a highdegree of selectivity, relatively stringent conditions are used to formthe duplexes, such as low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of between about50° C. to 70° C. For some applications, less stringent hybridizationconditions are required such as 0.15 M to 0.9 M salt; at temperaturesranging from between about 20° C. to 55° C. Hybridization conditions canalso be rendered more stringent by the addition of increasing amounts offormamide, to destabilize the hybrid duplex. Thus, particularhybridization conditions can be readily manipulated, and will generallybe a method of choice depending on the desired results. In general,convenient hybridization temperatures in the presence of 50% formamideare: 42° C. for a probe which is 95 to 100% homologous to the targetfragment, 37° C. for 90 to 95% homology and 32° C. for 85 to 90%homology.

[0117] In a clinical diagnostic embodiment, the nucleic acid sequencesof the about 200 kDa protein genes of the present invention may be usedin combination with an appropriate means, such as a label, fordetermining hybridization. A wide variety of appropriate indicator meansare known in the art, including radioactive, enzymatic or other ligands,such as avidin/biotin and digoxigenin-labelling, which are capable ofproviding a detectable signal. In some diagnostic embodiments, an enzymetag such as urease, alkaline phosphatase or peroxidase, instead of aradioactive tag may be used. In the case of enzyme tags, colorimetricindicator substrates are known which can be employed to provide a meansvisible to the human eye or spectrophotometrically, to identify specifichybridization with samples containing about 200 kDa protein genesequences.

[0118] The nucleic acid sequences of the about 200 kDa protein genes ofthe present invention are useful as hybridization probes in solutionhybridizations and in embodiments employing solid-phase procedures. Inembodiments involving solid-phase procedures, the test DNA (or RNA) fromsamples, such as clinical samples, including exudates, body fluids (e.g., serum, amniotic fluid, middle ear effusion, sputum, bronchoalveolarlavage fluid) or even tissues, is adsorbed or otherwise affixed to aselected matrix or surface. The fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes comprisingthe nucleic acid sequences of the about 200 kDa protein encoding genesor fragments or analogs thereof of the present invention under desiredconditions. The selected conditions will depend on the particularcircumstances based on the particular criteria required depending on,for example, the G+C contents, type of target nucleic acid, source ofnucleic acid, size of hybridization probe etc. Following washing of thehybridization surface so as to remove non-specifically bound probemolecules, specific hybridization is detected, or even quantified, bymeans of the label. It is preferred to select nucleic acid sequenceportions which are conserved among species of Moraxella. The selectedprobe may be at least 18bp and may be in the range of about 30 to 90 bp.

[0119] 4. Expression of the about 200 kDa Protein Gene

[0120] Plasmid vectors containing replicon and control sequences whichare derived from species compatible with the host cell may be used forthe expression of the genes encoding the about 200 kDa protein inexpression systems. The vector ordinarily carries a replication site, aswell as marking sequences which are capable of providing phenotypicselection in transformed cells. For example, E. coli may be transformedusing pBR322 which contains genes for ampicillin and tetracyclineresistance and thus provides easy means for identifying transformedcells. The plasmids or phage, must also contain, or be modified tocontain, promoters which can be used by the host cell for expression ofits own proteins.

[0121] In addition, phage vectors containing replicon and controlsequences that are compatible with the host can be used as atransforming vector in connection with these hosts. For example, thephage in lambda GEM™-11 may be utilized in making recombinant phagevectors which can be used to transform host cells, such as E. coliLE392.

[0122] Promoters commonly used in recombinant DNA construction includethe β-lactamase (penicillinase) and lactose promoter systems and othermicrobial promoters, such as the T7 promoter system as described in U.S.Pat. No. 4,952,496. Details concerning the nucleotide sequences ofpromoters are known, enabling a skilled worker to ligate themfunctionally with genes. The particular promoter used will generally bea matter of choice depending upon the desired results. Hosts that areappropriate for expression of the about 200 kDa protein genes,fragments, analogs or variants thereof, may include E. coli, Bacillusspecies, Haemophilus, fungi, yeast, Bordetella, or the baculovirusexpression system may be used.

[0123] In accordance with this invention, it is preferred to make theprotein by recombinant methods, particularly when the naturallyoccurring about 200 kDa protein as purified from a culture of a speciesof Moraxella may include trace amounts of toxic materials or othercontaminants. This problem can be avoided by using recombinantlyproduced protein in heterologous systems which can be isolated from thehost in a manner to minimize contaminants in the purified material.Particularly desirable hosts for expression in this regard include Grampositive bacteria which do not have LPS and are, therefore, endotoxinfree. Such hosts include species of Bacillus and may be particularlyuseful for the production of non-pyrogenic about 200 kDa protein,fragments or analogs thereof.

[0124] Biological Deposits

[0125] Certain plasmids that contain portions of the gene having theopen reading frame of the gene encoding the about 200 kDa outer membraneprotein of M. catarrhalis strain 4223 that are described and referred toherein have been deposited with the America Type Culture Collection(ATCC) located at 12301 Parklawn Drive, Rockville, Md., 20852, U.S.A.,pursuant to the Budapest Treaty and pursuant to 37 CFR 1.808 and priorto the filing of this application. The identifications of the respectiveportions of the gene present in these plasmids are shown in FIG. 5.

[0126] Samples of the deposited plasmids will become available to thepublic upon grant of a patent based upon this United States patentapplication. The invention described and claimed herein is not to belimited in scope by plasmids deposited, since the deposited embodimentis intended only as an illustration of the invention. Any equivalent orsimilar plasmids that encode similar or equivalent antigens as describedin this application are within the scope of the invention. Plasmid ATCCDesignation Date Deposited pKS47 97,111 Apr. 7, 1995 pKS5 97,110 Apr. 7,1995 pKS9 97,114 Apr. 18, 1995 

EXAMPLES

[0127] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific Examples. These Examples are described solely forpurposes of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitations.

[0128] Methods of molecular genetics, protein biochemistry,, andimmunology used but not explicitly described in this disclosure andthese Examples are amply reported in the scientific literature and arewell within the ability of those skilled in the art.

Example 1

[0129] This Example illustrates the generation of a non-clumping strain(RH408) of M. catarrhalis.

[0130]M. catarrhalis strain 4223, a clumping strain (a common propertyof Moraxella strains), was inoculated into several flasks containing 20mL of brain heat infusion (BHI) broth, and the cultures were incubatedwith shaking (170 rpm) overnight at 37° C. Five mL of each overnightculture were transferred to five individual 1 mL tubes, and were leftsitting undisturbed at room temperature for 3 to 8 hours, to allowbacteria to sediment. One hundred μL of the cleared upper phase of eachculture were used to inoculate 25 mL of BHI broth and cultures wereincubated overnight at 37° C., as described above. This passaging wasrepeated six times, using 25 μL of cleared culture to inoculate 25 mL ofBHI for each overnight culture. Non-clumping bacterial cultures wereidentified by measuring the absorbency A₅₇₈ at intervals over a 3 hourtime period, in order to compare the sedimentation rates of the passagedstrains to that of the original M. catarrhalis strain 4223 culture.Non-clumping mutants, including M. catarrhalis RH408, did not aggregateduring the three hour time period. On BHI agar plates, strain RH408 hada colony morphology typical for all non-clumping strains. Strain RH408was previously deposited in connection of U.S. application Ser. No.08/328,589 at the ATCC under the Budapest Treaty on Dec. 13, 1994 withAccession No. 55637.

Example 2

[0131] This Example illustrates the identification of the about 200 kDaouter membrane protein of Moraxella catarrhalis.

[0132]M. catarrhalis strains 4223, RH408, 5191, 8185, M2, M5, ATCC25240, 3, 56, 135, 585 were grown in brain heart infusion (BHI) broth.The culture was incubated overnight with aeration at 37° C.

[0133]M. catarrhalis cells were sonicated and total protein wasdetermined using the BCA assay system (Pierce, Rockford, Ill.). Ten μgof total protein were mixed with the SDS-PAGE sample buffer containing0.3M Tris-HCl (pH 8.0), 50% glycerol, 10% SDS, 20% β-mercaptoethanol and0.01% bromophenol blue, boiled for 5 minutes and loaded on each lane ofSDS-PAGE gel (0.75 mm thick, 7.5% acrylamide). The gels were run at 200V for 1 hour. Proteins were visualized by staining gels with a solutioncontaining 0.13% Coomassie brilliant blue, 10% acetic acid and 45%methanol. Excess stain was removed with a destaining solution of 5%ethanol and 7.5% acetic acid.

[0134] The various Moraxella proteins separated by this procedure areshown in FIGS. 1A and 1B. The M. catarrhalis strains tested were asfollows: FIG. 1A Lane Bacterial Strain Source 1. Molecular WeightStandards 2. E. coli 3. No sample 4. M. catarrhalis 4223 middle earfluid 5. M. catarrhalis RH408 non-clumping variant of 4223 6. M.catarrhalis 5191 middle ear fluid 7. M. catarrhalis 8185 nasopharynx 8.M. catarrhalis M2 sputum 9. M. catarrhalis M5 sputum 10. M. catarrhalis25240 ATCC 25240

[0135] FIG. 1B Lane Bacterial Strain Source 1. E. coli 2. No sample 3.Molecular Weight Size Markers 4. M. catarrhalis 4223 middle ear fluid 5.M. catarrhalis RH408 non-clumping variant of 4223 6. M. catarrhalis 3sputum 7. M. catarrhalis 56 sputum 8. M. catarrhalis 135 middle earfluid 9. M. catarrhalis 585 Blood

[0136] The about 200 kDa outer membrane protein was clearly seen in allotitis media strains (M. catarrhalis 4223, 5191, 135), in one strainisolated from the nasopharynx (8185), and in one strain isolated fromsputum (M2). However, the about 200 kDa protein was not detected inthree isolates from sputum (3, 56 and M5) and in one strain with unknownorigin (ATCC 25240). A very narrow band was found in an isolate fromblood of a bacteremia patient (585) and this band was recognized by ananti-200 kDa specific guinea pig serum on an immunoblot. Strain RH408 isa non-clumping spontaneous mutant isolated from strain 4223 (seeExample 1) and was found to not express the about 200 kDa protein.

[0137] When gels were run longer, they showed heterogeneity in theapparent molecular masses of the about 200 kDa outer membrane protein indifferent strains of M. catarrhalis (FIG. 2). In FIG. 2 the strainsanalyzed were as follows: Lane Strain Source 1. Molecular Weight SizeMarkers 2. M. catarrhalis H04 middle ear fluid 3. M. catarrhalis H12middle ear fluid 4. M. catarrhalis PO34 middle ear fluid 5. M.catarrhalis PO51 middle ear fluid 6. M. catarrhalis E-07 middle earfluid 7. M. catarrhalis E-22 middle ear fluid 8. M. catarrhalis E-23middle ear fluid 9. M. catarrhalis RH 4223 middle ear fluid 10. M.catarrhalis RH 408 Non-clumping variant of 4223

[0138] The strain H12 (lane 3) was a natural isolate from middle earfluid, but did not produce the about 200 kDa protein.

[0139] There may be at least three different sizes of protein in theabout 200 kDa range. However, antibodies raised against the about 200kDa outer membrane protein from one strain of M. catarrhalis (4223) didrecognize all about 200 kDa proteins tested, present in differentstrains of M. catarrhalis. It is possible, however, that in particularimmunogenic compositions, for example, as a vaccine and in particulardiagnostic embodiments, that the about 200 kDa outer membrane proteinfrom a variety of M. catarrhalis isolates (including immunogenicallydiverse isolates) may be required.

Example 3

[0140] This Example illustrates the detection of antibodies specific forthe about 200 kDa outer membrane protein in a serum obtained from aconvalescent patient having recovered from otitis media due to M.catarrhalis.

[0141] After separation by SDS-PAGE, bacterial proteins were transferredfrom polyacrylamide gels to prepared PVDF (polyvinylidene fluoride;Millipore) membranes at a constant voltage of 70 V for 1.5 h in a buffersystem consisting of 3 g Tris, 14,4 g glycine and 200 ml methanol perliter at 4° C. Membranes with transferred proteins were blocked withBlocking Reagent (from Boehringer Mannheim) diluted in TBS (0.1 M Tris,0.15M Nacl) at room temperature for 30 min. Blots were exposed toconvalescent antiserum diluted 1:500 in Blocking Reagent/TBS with 0.1%Tween 20 for 2 hours at room temperature. This patient had otitis mediaand the M. catarrhalis strain isolated from the patient's ear fluid wasM. catarrhalis CJ7. Blots were then washed 2 times in BlockingReagent/TBS with Tween at 15 min per wash. The reporter conjugate,horseradish peroxidase (HRP) conjugated to protein G, was diluted 1:4000with Blocking Reagent/TBS with Tween and used to immerse the washedmembranes for 30 min at room temperature. Blots were washed twice asabove, followed by a TBS wash. Bound antibodies were detected using theLumiGlo (Kirkegaard and Perry) chemiluminescent detection system asdescribed by the manufacturer. Treated blots were exposed to X-ray film.Antibodies were detected in this convalescent serum that reacted withthe about 200 kDa outer membrane protein of M. catarrhalis CJ7. Theseresults indicate that the about 200 kDa outer membrane protein is animmunogenic protein of M. catarrhalis to which an immune response iselicited during a natural infection by M. catarrhalis.

Example 4

[0142] This Example illustrates the isolation and purification of theabout 200 kDa outer membrane protein.

[0143]M. catarrhalis 4223 cells were harvested by centrifugation at2,000 rpm for 10 min and frozen. The frozen cells were thawed,resuspended in 20 mM sodium -phosphate buffer (pH 7.2) and sonicateduntil the cells were disrupted. The frozen-thawed cells were also lysedin 20 mM Tris buffer (pH 8) containing 4% SDS and 0.2 mM EDTA by boilingfor 5 min to produce a cell lysate. The cell sonicates and cell lysateswere suspended in a SDS-polyacrylamide gel electrophoresis (SDS-PAGE)sample buffer, boiled for 5 min and separated by SDS-PAGE on a gel (1.5mm thick, 7.5% acrylamide). The estimated position of the about 200 kDaprotein on the gel was cut out and the protein extracted from the gel byelectroelution using the same buffer as the SDS-PAGE running buffer. Theisolated about 200 kDa outer membrane protein was shown to be ahomogeneous, single band by SDS-PAGE as seen in FIG. 3. The samplesanalyzed in FIG. 3 are as follows: Lane Sample 1. Molecular Weight SizeMarkers 2. Isolated and purified 200 kDa outer membrane protein

[0144] The isolated and purified 200 kDa outer membrane protein of M.catarrhalis shown in FIG. 3 has a purity of at least 70%. Purified about200 kDa outer membrane protein preparations of at least 95% could bereadily achieved.

Example 5

[0145] This Example illustrates the immunization of guinea pigs withpurified about 200 kDa protein from M. catarrhalis.

[0146] Approximately 30 to 40 μg of, the about 200 kDa protein, whichwas isolated from M. catarrhalis strain 4223 by electroelution, weremixed with Freund's complete adjuvant (FCA) and subcutaneously injectedinto guinea pigs. After two weeks, the animals were boosted with aboutthe same amount of the about 200 kDa protein in incomplete Freund'sadjuvant (IFA). Two weeks later, blood was collected from the guineapigs and antisera were obtained.

[0147] One antiserum was examined on Western blot for its reactivitywith the about 200 kDa protein present in 54 different strains of M.catarrhalis, which were isolated in different geographical locationsthroughout the world (Canada, U.S. and Finland) (see Table 1 below). Theabout 200 kDa protein was recognized by the antiserum in all strains, inwhich the presence of the about 200 kDa protein band was detected onSDS-PAGE gels stained with Coomassie Blue. These results indicate thatcommon epitopes of the about 200 kDa protein were present in all M.catarrhalis strains, which possessed this protein. As stated earlier,this protein is not present in all M. catarrhalis strains, but almostall strains, which were isolated from middle ear fluids from otitismedia patients, did possess this protein (Table 1).

Example 6

[0148] This Example illustrates the specific recognition of M.catarrhalis strain 4223 with anti-200 kDa protein guinea pig serum byELISA assay (see Table 2 below).

[0149]M. catarrhalis strains 4223, RH408 (200 kDa protein negativemutant) and H-12 were cultured in 60 mL of BHI broth overnight. E. colistrain BL21 (DE3) was cultured in 60 mL of broth overnight. The cultureswere split into three tubes and centrifuged. M. catarrhalis strain 4223was centrifuged at 1,500 rpm for 10 min., H-12 at 2,000 rpm for 10 min.,and RH408 and E. coli BL21 (DE3) at 3,000 rpm for 15 min. The pellet inone tube was suspended in 20 ml of Dulbecco's phosphate buffered saline(D-PBS) and diluted to 1/500 with coating buffer (0.05Mcarbonate/bicarbonate buffer) pH 9.6. One hundred μL of the bacteriasuspension were placed in each well and incubated for 1 hour at roomtemperature. One hundred μL of 0.2% glutaraldehyde was added to eachwell and incubated at room temperature for 10 min. to fix the cells onthe well. The wells were washed with PBS containing 0.1% Tween 20 and0.1% BSA (washing buffer), and then blocked with PBS containing 0.1% BSAfor 30 min. at room temperature. After washing 5 times for 10 secondswith the washing buffer, serial dilutions of guinea pig antiserum withthe washing buffer were added to the wells and incubation at roomtemperature was continued for 60 min. After washing, goat anti-guineapig IgG conjugated with horseradish peroxidase was added to each well atthe dilution of 1/20,000. After incubation at room temperature for 60minutes, the wells were washed and then color reaction wasdeveloped-using 3,3-5,15-tetramethylbenzidene (TMB) and hydrogenperoxide.

[0150] The ELISA plate wells were also coated with sonicates containing10 μg/mL of total proteins in the coating buffer, blocked without thefixation process and then assayed as described above.

[0151] The results shown in Table 2 indicate that the about 200 kDaouter membrane protein specific guinea pig antiserum specificallyrecognizes strains of M. catarrhalis which produce the about 200 kDaprotein. The ability of the antiserum to recognize whole cells indicatesthat the protein is present on the surface of the bacterial cells.

Example 7

[0152] This Example describes the determination of an internal aminoacid sequence of the 200 kDa outer membrane protein.

[0153] The about 200 kDa outer membrane protein was isolated from M.catarrhalis 4223 by electroelution as described above. The protein wassubjected to CNBr degradation, the proteolytic digests subjected toSDS-PAGE and transferred onto PVDF membrane. A peptide band migrating ata position corresponding to approximately 40 kDa was cut out from themembrane and its N-terminal amino acid sequence was determined. Inanother experiment, the CNBr degradation products of the about 200 kDaprotein were subjected to a direct determination of N-terminal aminoacid sequencing without separating by SDS-PAGE. Both analyses gave anidentical, N-terminal sequence of 20 amino acids with one unidentifiedamino acid at the 17th position. The internal sequence of the 200 kDaouter membrane protein was:

[0154]NH₂-Asn-Val-Lys-Ser-Val-lle-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-X-Gln-Gly-lle(SEQ ID No: 5).

Example 8

[0155] This Example describes the immunization of guinea pigs with apeptide corresponding to an internal fragment of the about 200 kDa outermembrane protein and the analysis of the antiserum generated.

[0156] Based upon the determination of the amino acid sequence of aninternal fragment of the about 200 kDa outer membrane protein asdescribed above, a 16 amino acid long peptide of sequence:

[0157]NH₂-Asn-Val-Lys-Ser-Val-lle-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQID No: 6) was synthesized using standard procedures. This 16-mer peptidewas conjugated to KLH using Imject Maleimide Activated KLH (Pierce,Rockford, Ill.) and approximately 500 μg of the conjugate was injectedinto guinea pigs using the same immunization and boosting schedule asdescribed above. The guinea pig anti-serum raised against the 16-merinternal amino acid sequence (SEQ ID No: 6) was examined by immunoblotanalysis and found to specifically recognize 200 kDa outer membraneprotein in cell sonicates of M. catarrhalis 4223. The results are shownin FIG. 4 and indicate that the anti-peptide guinea pig antiserumspecifically recognizes the 200 kDa protein of M. catarrhalis 4223. Thesamples analyzed in FIG. 4 were as follows: Lane Sample Antiserum 1.Molecular Weight Markers 2. Purified 200 kDa outer membrane Anti-200 kDaprotein protein 3. M. catarrhalis cell sonicate Anti-peptide 1:5000 4.M. catarrhalis cell sonicate Anti-peptide 1:1000 5. M. catarrhalis cellsonicate Anti-peptide 1:500 6. M. catarrhalis cell sonicate Pre-immuneserum

[0158] The results obtained confirm that the peptide corresponding toSEQ ID Nos: 5 and 6 are derived from the 200 kDa outer membrane protein.

Example 9

[0159] This Example describes the preparation of a M. catarrhalisgenomic library.

[0160] Chromosomal DNA was isolated as follows:

[0161] An M. catarrhalis cell pellet was resuspended in 20 mL ofTris-EDTA (TE) buffer, pH 7.5. Pronase (final concentration 500 μg/mL)and SDS (final concentration 1%) were added and the suspension wasincubated at 37° C. for 2 hours. DNA was isolated by sequentialextractions once with phenol, twice with phenol-chloroform (1:1), andonce with chloroform-isoamyl alcohol (24:1). Extracted DNA was dialyzedagainst 1M NaCl at 4° C. for 4 hours. This was followed by dialysisagainst TE buffer, pH 7.5, at 4° C. for 48 hours (3 buffer changes). DNAwas ethanol precipitated from the dialysate. Large-size DNA wascollected by spooling on a glass rod, air dried and dissolved in 3 mLwater. Small scale Sau3A (New England BioLabs) restriction digests ofchromosomal DNA (final volume 10 μl) were done to establish conditionsrequired to obtain maximal amounts of chromosomal DNA with a size rangeof 15-23 kb. Large scale digests were prepared once the optimaldigestion conditions were determined. The large scale digests consistedof 50 μL of chromosomal DNA (290 μg/mL), 33 μL water, 10 μL Sau3A buffer(New England BioLabs), 1 μL BSA (10 mg/ml, New England BioLabs) and 6.3μL Sau3A (0.04 U/μL), and were incubated at 37° C. for 15 min. Reactionswere stopped by the addition of 10 μL 10× loading buffer (100 mMTris-HCl pH 8, 10 mM EDTA, 0.1% bromophenol blue, 50% glycerol).Digested DNA was applied to 0.5% agarose gels (prepared inTris-acetate-EDTA (TAE)) and separated according to size at 50 V for 6hours. The region of the gel encompassing DNA of size 15-23 kb was cutfrom the gel and placed in dialysis tubing (BRL) with 3 mL of TAE. DNAwas electroeluted from the gel-slice overnight at a field strength of 1V/cm. Electroeluted DNA in TAE was extracted once with phenol, once withphenol-chloroform (1:1), and precipitated with ethanol. The dried DNApellet was dissolved in 5 AL water. Size-fractionated chromosomal DNAwas ligated with BamHI cut EMBL3 arms (Promega) using T4 DNA ligase in afinal volume of 9 μL. The entire ligation reaction was packaged intophage λ using a commercial packaging kit (Amersham) following themanufacturer's protocol.

[0162] The packaged DNA library was amplified on solid medium. This wasaccomplished by incubating 0.1 ml E. coli strain NM539 plating cellssuspended in 10 mM MgSO₄ with 15-25 μL of the packaged DNA library at37° C. for 5 minutes. Bacteria with adsorbed phage were plated onto BBLplates (10 g BBL trypticase peptone, 5 g NaCl and 15 g agar per litre)using 3 mL of BBL top-agarose (same as BBL plates except agar replacedwith 0.6% agarose) and plates were incubated overnight at 37° C. Phagewere eluted from the top-agarose by adding 3 mL SM buffer (50 mMTris-HCl, pH 7.5, 8 mM MgSO₄, 100 mM NaCl, 0.01% gelatin) to the platesand leaving them at 4° C. for 7 hours. SM buffer containing phage wascollected from the plates, transferred to a screwcap tube and stored at4° C. over chloroform.

Example 10

[0163] This Example describes the cloning of a gene encoding the M.catarrhalis 200 kDa outer membrane protein.

[0164] The M. catarrhalis genomic library in phage lambda EMBL3 wasscreened using an anti-200 kDa protein guinea pig antiserum. A lambdaphage clone 8II, which expressed an about 200 kDa protein, was confirmedby immunoblotting of the phage lysate using the about 200 kDa outermembrane-specific antiserum.

[0165] Plate lysate cultures of this recombinant phage were prepared.The DNA was extracted from the plate lysates using a Wizard Lambda PrepsDNA Purification System (Promega Corp, Madison, Wis.) according to themanufacturer's instructions. This phage clone carried a DNA insert ofabout 16 kb in size (the restriction map for which is shown in FIG. 5).The phage DNA was digested with a mixture of the restriction enzymesSalI and XhoI, and separated by agarose gel electrophoresis. Two DNAbands, approximately 5 kb and 11 kb in size, respectively, were cut outfrom the gel and extracted using a Geneclean kit (BIO 101 Inc., LaJolla,Calif.) according to the manufacturer's direction.

[0166] The smaller 5 kb fragment was ligated into a plasmid vector,pBluescript II SK ± (Stratagene Cloning Systems, LaJolla, Calif.), whichhad been previously digested with SalI and XhoI, to produce plasmidpKS5. The larger 11 kb fragment was ligated into a plasmid vector, pSP72(Promega Corp., Madison, Wis.), to produce plasmid pKS9. Both ligatedplasmids were used to transform E. coli, strain DH5α.

[0167] The lambda phage DNA was also digested with a mixture of XhoI andKpnI and the approximately 1.2 kb fragment was isolated after agarosegel separation as described above. This 1.2 kb fragment was ligated intoa plasmid vector, pGEM-7Zf(+) (Promega Corp., Madison, Wis.), to produceplasmid pKS47. Restriction maps of the plasmid clones are shown in FIG.5.

Example 11

[0168] This Example describes the sequencing of the gene encoding theabout 200 kDa outer membrane protein of M. catarrhalis.

[0169] The gene encoding the about 200 kDa outer membrane protein wassequenced using an Applied Biosystems sequencer. The one strand of theinsert in the plasmid pKS5, was sequenced after construction of a nestedset of deletions using a Erase-a-Base system (Promega Corp., Madison,Wis.). The plasmid pKS5 was first digested with XhoI and KpnI, treatedwith exonuclease III to generate a nested set of deletions in the insertand then recircularized according to the manufacturer's directions. E.coli DH5α was transformed with a series of plasmids with deletionsgenerated in this way. Plasmids were isolated from the transformantsusing a Quiagen midi plasmid isolation kit (Qiagen) and the size ofplasmids examined by agarose gel electrophoresis after restrictionenzyme digestion. The inserts of the plasmids with deletions weresequenced using a bacteriophage T7 promoter sequence as a primer.

[0170] Based upon the sequence, nucleotide primers were synthesized.Using the synthetic nucleotide primers, sequence gaps, which were notsequenced by the Erase-a Base system, were determined.

[0171] The sequences of the inserts in plasmids pKS47 and pKS71 weredetermined from both ends using synthetic nucleotide primers. Thenucleotide sequence of the gene has an open reading frame of the genecoding for the about 200 kDa outer membrane protein of M. catarrhalis asshown in FIG. 6 (SEQ ID No: 2). This sequence included a nucleotidesequence:

[0172] 5′-AATGTCAAATCAGTCATTAACAAAGAACAAGTAAATGATGCCAATAAAAAGCAAGGCATC-3′ (SEQ ID No: 9)

[0173] which encodes the internal amino acid sequence of the about 200kDa outer membrane protein (SEQ ID No: 5) determined above. This resultconfirms that the cloned gene has an open reading frame of the genecoding for the about 200 kDa outer membrane protein of M. catarrhalis.The gene encodes a protein having 1992 amino acids, a calculatedmolecular weight of 204,677 and a calculated amino acid composition asshown in Table III below. The deduced amino acid sequence of the proteinis shown in FIG. 6 (SEQ ID No: 3).

Example 12

[0174] This Example describes the identification of the start codon ofthe gene encoding the about 200 kDa gene of M. catarrhalis.

[0175] To identify the translation start codon and the promoter regionof the 200 kDa protein gene, a plasmid, pKS80, was constructed from pKS5and pKS47 (FIG. 5). This construct contained about 250 base pairs of DNAupstream from the ATG. The plasmid, pKS5, was digested with KpnI andXhoI. The digest was separated on 0.8% agarose gel and the about 8 kbDNA fragment was cut out from the gel and extracted. Another plasmid,pKS47, was also digested with the two enzymes and the about 1.1 kb DNAfragment was extracted. The 1.1 kb fragment was ligated to the 8 kbfragment to construct pKS80. Western blots using anti-200 kD proteinguinea pig serum failed to detect 200 kD protein in the lysates of thetransformants carrying PKS80.

[0176] To examine if the construct was too long to be expressed in E.coli, three different sizes of C-terminal truncations were constructed,as shown in FIG. 8. First, the whole insert in pKS80 was cut out bydigestion with KpnI and BamHI and then inserted into another vectorplasmid, pGEM7Zf(+) (Promega, Madison Wis.), which had been previouslydigested with the same two enzymes. The resulting plasmid, pKS105, wasfurther digested with either one of the following enzymes, (1) HindIII,(2) HpaI and SmaI or (3) EcoRV, gel-purified and then recircularized toproduce pKS130, pKS136 and pKS144, respectively. Transformants of E.coli, DH5α, with either one of pKS130, pKS136 or pKS144 did not produceany truncated proteins, when examined on Western blots using anti-200 kDprotein guinea pig serum.

[0177] Next, to investigate if the start codon was GTG and if thepromoter region was further upstream from the GTG, an about 0.9 kbfragment was cut out from pKS71 using ApaI and KpnI, and ligated intopKS130, pKS136 and pKS144, which had been previously digested with ApaIand KpnI. The 0.9kb fragment from pKS71 carried the NcoI-KpnI fragment,which contained the possible start codon, GTG, and about 700 bp upstreamregion from the GTG (FIG. 8). The resulting constructs, pKS159, pKS149and pKS155, produced truncated proteins, which were recognized byanti-200 kDa protein guinea pig serum on Western blots. The ApaI andKpnI fragment was also ligated to pKS105, which had no C-terminaltruncation, to produce pKS164. The transformants carrying pKS164produced a full-length 200 kDa protein, which was recognized by the sameantiserum on Western blot. These results show that the 5′-region of thegene containing the GTG codon and its upstream sequence is necessary forexpression of the about 200 kDa protein gene from its own promoter in E.coli, and indicate that a translation start codon of the about 200 kDaprotein gene is GTG.

[0178] To confirm that the start codon of the gene is GTG, two peptideswere synthesized, as shown in FIG. 9, according to the deduced aminoacid sequence from the nucleotide sequence in FIG. 6. Peptide 1 (SEQ IDNo: 12) encompasses the 30 amino acids from the GTG start codon. Peptide2 (SEQ ID No: 12) is the next 30 amino acid peptide. The peptides areidentified in FIG. 6 by underlining. Antisera were raised against thesetwo peptides in guinea pigs and antisera were obtained. As seen in FIG.10, antisera raised against these two peptides clearly recognized 200kDa protein from M. catarrhalis, strain 4223, by Western blotting. M.catarrhalis, strain 4223, was sonicated. Proteins in the sonicate wereseparated on a SDS-PAGE gel and transferred to PVDF membrane. Themembrane was cut into strips and treated with either anti-peptide 1 oranti-peptide 2 guinea pig serum as a first antibody. The second antibodywas goat anti-guinea pig IgG conjugated with horse radish peroxidase(Jackson ImmunoResearch Lab. Inc., West Grove, Pa.). The membrane wasfinally treated with CN/DAB substrate (Pierce, Rockford, Ill.) for colordevelopment. Lane 1: prestained molecular weight marker, Lane 2:anti-200 kD protein serum, Lane 3: anti-peptide I serum from guinea pigNo. 1, Lane 4: prebleed serum from guinea pig No. 1, Lane 5:anti-peptide 1 serum from guinea pig No. 2, Lane 6: prebleed serum fromguinea pig No. 2, Lane 7: anti-peptide 2 serum from guinea pig No. 3,Lane 8: prebleed serum from guinea pig No. 3, Lane 9: anti-peptide 2serum from guinea pig No. 4, Lane 10: prebleed serum from guinea pig No.4. The results shown in FIG. 10 indicate that the GTG is the translationstart codon of the gene encoding the about 200 kDa protein.

[0179] The coding sequence of the about 200 kDa protein gene, whichstarts at GTG, is 5976 bp and encodes a protein of 1992 amino acids anda calculated molecular weight of 204,677. The position of the 200 kDaprotein gene is shown in FIG. 5. The sequence between NcoI and SalI andits amino acid translation are shown in FIG. 6. The calculated aminoacid composition of the about 200 kDa protein is shown in Table III.

[0180] To construct two different sizes of N-terminal truncation genesunder the control of the T7 promoter (as shown in FIG. 11), a ScaI-SalIfragment, which carried the about 1.9kb 3′-region of the about 200 kDaprotein gene, was cut out from pKS5, and the PvuII-SalI fragment, whichcarried the about 4.8 kb 3′-region, was cut out from pKS80. The twofragments were ligated into a plasmid, pT7-7, previously digested withSmaI and SalI, to produce pKS94 and pKS91, respectively. These ligationsresulted in fusions of 1.9 kb and 4.8 kb 3′-regions with sevenN-terminal amino acids from the vector. When transformants of an E. colistrain, BL21(DE3)/pLysS, with either pKS94 or pKS91 were induced withIPTG, they produced a large quantity of N-terminally truncated 200 kDaprotein. FIG. 12 shows a Western blot showing the expression of thetruncated protein by one of transformants carrying the pKS94 plasmid.

[0181] A LacZ fusion of the 3′-5.5 kb fragment of the about 200 kDaprotein gene, as shown in FIG. 11. The 5.8 kb fragment, which containedthe 3′-5.5 kb region of about 200 kDa protein gene, was excised frompKS80 by digestion with PstI, gel-purified, and then ligated topGEM5Zf(+) (Promega, Madison, Wis.), previously digested with the sameenzyme. The E. coli DH5α clones, which carried the gene in the samedirection and reading frame as the LacZ α peptide, were selected byrestriction enzyme analyses. These clones constitutively expressed thefusion protein, as shown in FIG. 13.

SUMMARY OF THE DISCLOSURE

[0182] In summary of the disclosure, the present invention provides anisolated and purified outer membrane protein of a Moraxella strain,particularly M. catarrhalis, having a molecular weight of about 200 kDaas well as isolated and purified DNA molecules encoding the outermembrane protein. The invention also provides analogs, truncations andpeptides corresponding to portions of the outer membrane protein. Theprotein, DNA sequences, recombinant proteins derived therefrom andpeptides are useful for diagnosis, immunization and the generation ofdiagnostic and immunological reagents. Modifications are possible withinthe scope of this invention. TABLE I Presence of the about 200 kDa outermembrane protein in various isolates of Moraxella catarrhalis Number ofisolates¹ Type of Clinical Number of isolates containing the 200 kDaIsolate Examined outer membrane protein Otitis Media 37 36Sputum/Expectoration/ 13 6 Bronchial Secretion Blood 2 2 Nasopharynx 1 1Unknown 1 0

[0183] TABLE II Detection of about 200 kDa outer membrane protein of M.catarrhalis by the monospecific anti-200 kDa outer membrane guinea pigantiserum Strain Sample Reciprocal Reactive Titre 4223 Whole cells notfixed 800 RH408 Whole cells not fixed <200 H12 Whole cells not fixed<200 E. coli BL21 Whole cells not fixed <200 4223 Whole cells fixed 3200RH408 Whole cells fixed 200 H12 Whole cells fixed <200 E. coli BL21Whole cells fixed <200 4223 Sonicate 12,800 RH408 Sonicate 800 H12Sonicate 800 E. coli BL21 Sonicate 200

[0184] TABLE III Amino acid composition of the about 200 kDa outermembrane protein of M. catarrhalis Residue Number Percentage (MW) N -Asparagine 196 10.9 T - Threonine 221 10.9 K - Lysine 159 10.0 D -Aspartic Acid 147 8.3 A - Alanine 219 7.6 V - Valine 148 7.2 I -Isoleucine 116 6.4 S - Serine 150 6.4 G - Glycine 222 6.2 L - Leucine111 6.1 Q - Glutamine 83 5.2 E - Glutamic Acid 55 3.5 F - Phenylalanine40 2.9 R - Arginine 34 2.6 Y - Tyrosine 27 2.2 H - Histidine 24 1.6 P -Proline 30 1.4 M - Methionine 7 .4 W - Tryptophan 3 .3 B - Aspartic AcidAsparagine 0 .0 C - Cysteine 0 .0

REFERENCES

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1 10 1 6973 DNA Moraxella catarrhalis CDS (708)..(6683) 1 ccatggatatgggcaggtgt gctcgcctgc cgtatgatgg cgatgacacc ccatttgccc 60 catatctgtacgatttgaca tgtgatatga tttaacatgt gacatgattt aacattgttt 120 aatactgttgccatcattac cataatttag taacgcattt agtaacgcat ttgtaaaaat 180 cattgcgcccctttatgtgt atcatatgaa tagaatatta tgattgtatc tgattattgt 240 atcagaatggtgatgctata tgatgatgcc tacgagttga tttgggttaa tcactctatg 300 atttgatatattttgaaact aatctattga cttaaatcac catatggtta taatttagca 360 taatggtaggctttttgtaa aaatcacatc gcaatattgt tctactgtta ctaccatgct 420 tgaatgacgatcccaatcac cagattcatt caagtgatgt gtttgtatac gcaccattta 480 ccctaattatttcaatcaaa tgcctatgtc agcatgtatc atttttttaa ggtaaaccac 540 catgaatcacatctataaag tcatctttaa caaagccaca ggcacattta tggcagtggc 600 agagtacgccaaatcccaca gcacgggggg ggggtagctg tgctacaggg caagttggca 660 gtgtatgcactctgagcttt gcccgtattg ccgcgctcgc tgtcctc gtg atc ggt 716 Met Ile Gly 1gca acg ctc agt ggc agt gct tat gct caa aaa aaa gat acc aaa cat 764 AlaThr Leu Ser Gly Ser Ala Tyr Ala Gln Lys Lys Asp Thr Lys His 5 10 15 atcgca att ggt gaa caa aac cag cca aga cgc tca ggc act gcc aag 812 Ile AlaIle Gly Glu Gln Asn Gln Pro Arg Arg Ser Gly Thr Ala Lys 20 25 30 35 gcggac ggt gat cga gcc att gct att ggt gaa aat gct aac gca cag 860 Ala AspGly Asp Arg Ala Ile Ala Ile Gly Glu Asn Ala Asn Ala Gln 40 45 50 ggc ggtcaa gcc atc gcc atc ggt agt agt aat aaa act gtc aat gga 908 Gly Gly GlnAla Ile Ala Ile Gly Ser Ser Asn Lys Thr Val Asn Gly 55 60 65 agc agt ttggat aag ata ggt acc gat gct acg ggt caa gag tcc atc 956 Ser Ser Leu AspLys Ile Gly Thr Asp Ala Thr Gly Gln Glu Ser Ile 70 75 80 gcc atc ggt ggtgat gta aag gct agt ggt gat gcc tcg att gcc atc 1004 Ala Ile Gly Gly AspVal Lys Ala Ser Gly Asp Ala Ser Ile Ala Ile 85 90 95 ggt agt gat gac ttacat ttg ctt gat cag cat ggt aat cct aaa cat 1052 Gly Ser Asp Asp Leu HisLeu Leu Asp Gln His Gly Asn Pro Lys His 100 105 110 115 ccg aaa ggt actctg att aac gat ctt att aac ggc cat gca gta tta 1100 Pro Lys Gly Thr LeuIle Asn Asp Leu Ile Asn Gly His Ala Val Leu 120 125 130 aaa gaa ata cgaagc tca aag gat aat gat gta aaa tat aga cgc aca 1148 Lys Glu Ile Arg SerSer Lys Asp Asn Asp Val Lys Tyr Arg Arg Thr 135 140 145 acc gca agc ggacac gcc agt act gca gtg gga gcc atg tca tat gca 1196 Thr Ala Ser Gly HisAla Ser Thr Ala Val Gly Ala Met Ser Tyr Ala 150 155 160 cag ggt cat ttttcc aac gcc ttt ggt aca cgg gca aca gct aaa agt 1244 Gln Gly His Phe SerAsn Ala Phe Gly Thr Arg Ala Thr Ala Lys Ser 165 170 175 gcc tat tcc ttggca gtg ggt ctt gcc gcc aca gcc gag ggc caa tct 1292 Ala Tyr Ser Leu AlaVal Gly Leu Ala Ala Thr Ala Glu Gly Gln Ser 180 185 190 195 aca atc gctatt ggt tct gat gca aca tct agc tcg ttg gga gcg ata 1340 Thr Ile Ala IleGly Ser Asp Ala Thr Ser Ser Ser Leu Gly Ala Ile 200 205 210 gcc ctt ggtgca ggt act cgt gct cag cta cag ggc agt att gcc cta 1388 Ala Leu Gly AlaGly Thr Arg Ala Gln Leu Gln Gly Ser Ile Ala Leu 215 220 225 ggt caa ggttct gtt gtc act cag agt gat aat aat tct aga ccg gcc 1436 Gly Gln Gly SerVal Val Thr Gln Ser Asp Asn Asn Ser Arg Pro Ala 230 235 240 tat aca ccaaat acc cag gca cta gac ccc aag ttt caa gcc acc aat 1484 Tyr Thr Pro AsnThr Gln Ala Leu Asp Pro Lys Phe Gln Ala Thr Asn 245 250 255 aat acg aaggcg ggt cca ctt tcc att ggt agt aac tct atc aaa cgt 1532 Asn Thr Lys AlaGly Pro Leu Ser Ile Gly Ser Asn Ser Ile Lys Arg 260 265 270 275 aaa atcatc aat gtc ggt gca ggt gtt aat aaa acc gat gcg gtc aat 1580 Lys Ile IleAsn Val Gly Ala Gly Val Asn Lys Thr Asp Ala Val Asn 280 285 290 gtg gcacag cta gaa gcg gtg gtg aag tgg gct aag gag cgt aga att 1628 Val Ala GlnLeu Glu Ala Val Val Lys Trp Ala Lys Glu Arg Arg Ile 295 300 305 act tttcag ggt gat gat aac agt act gac gta aaa ata ggt ttg gat 1676 Thr Phe GlnGly Asp Asp Asn Ser Thr Asp Val Lys Ile Gly Leu Asp 310 315 320 aat acttta act att aaa ggt ggt gca gag acc aac gca tta acc gat 1724 Asn Thr LeuThr Ile Lys Gly Gly Ala Glu Thr Asn Ala Leu Thr Asp 325 330 335 aat aatatc ggt gtg gta aaa gag gct gat aat agt ggt ctg aaa gtt 1772 Asn Asn IleGly Val Val Lys Glu Ala Asp Asn Ser Gly Leu Lys Val 340 345 350 355 aaactt gct aaa act tta aac aat ctt act gag gtg aat aca act aca 1820 Lys LeuAla Lys Thr Leu Asn Asn Leu Thr Glu Val Asn Thr Thr Thr 360 365 370 ttaaat gcc aca acc aca gtt aag gta ggt agt agt agt agt act aca 1868 Leu AsnAla Thr Thr Thr Val Lys Val Gly Ser Ser Ser Ser Thr Thr 375 380 385 gctgaa tta ttg agt gat agt tta acc ttt acc cag ccc aat aca ggc 1916 Ala GluLeu Leu Ser Asp Ser Leu Thr Phe Thr Gln Pro Asn Thr Gly 390 395 400 agtcaa agc aca agc aaa acc gtc tat ggc gtt aat ggg gtg aag ttt 1964 Ser GlnSer Thr Ser Lys Thr Val Tyr Gly Val Asn Gly Val Lys Phe 405 410 415 actaat aat gca gaa aca aca gca gca atc ggc act act cgt att acc 2012 Thr AsnAsn Ala Glu Thr Thr Ala Ala Ile Gly Thr Thr Arg Ile Thr 420 425 430 435aga gat aaa att ggc ttt gct cga gat ggt gat gtt gat gaa aaa caa 2060 ArgAsp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val Asp Glu Lys Gln 440 445 450gca cca tat ttg gat aaa aaa caa ctt aaa gtg ggt agt gtt gca att 2108 AlaPro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly Ser Val Ala Ile 455 460 465acc ata gac aat ggc att gat gca ggt aat aaa aag atc agt aat ctt 2156 ThrIle Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Leu 470 475 480gcc aaa ggt agc agt gct aac gat gcg gtt acc atc gaa cag ctc aaa 2204 AlaLys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile Glu Gln Leu Lys 485 490 495gcc gcc aag cct act tta aac gca ggc gct ggc atc agt gtc aca cct 2252 AlaAla Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile Ser Val Thr Pro 500 505 510515 act gaa ata tca gtt gat gct aag agt ggc aat gtt acc gcc cca act 2300Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val Thr Ala Pro Thr 520 525530 tac aac att ggc gtg aaa acc acc gag ctt aac agt gat ggc act agt 2348Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser Asp Gly Thr Ser 535 540545 gat aaa ttt agt gtt aag ggt agt ggt acg aac aat agc tta gtt acc 2396Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn Ser Leu Val Thr 550 555560 gcc gaa cat ttg gca agc tat cta aat gaa gtc aat cga acg gct gac 2444Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn Arg Thr Ala Asp 565 570575 agt gct cta caa agc ttt acc gtt aaa gaa gaa gac gat gat gac gcc 2492Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp Asp Asp Asp Ala 580 585590 595 aac gct atc acc gtg gct aaa gat acg aca aaa aat gcc ggc gca gtc2540 Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn Ala Gly Ala Val 600605 610 agc atc tta aaa ctc aaa ggt aaa aac ggt cta acg gtt gct acc aaa2588 Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr Val Ala Thr Lys 615620 625 aaa gat ggt acg gtt acc ttt ggg ctt agc caa gat agc ggt ctg acc2636 Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp Ser Gly Leu Thr 630635 640 att ggc aaa agc acc cta aac aac gat ggc ttg act gtt aaa gat acc2684 Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr Val Lys Asp Thr 645650 655 aac gaa caa atc caa gtc ggt gct aat ggc att aaa ttt act aat gtg2732 Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys Phe Thr Asn Val 660665 670 675 aat ggt agt aat cca ggt act ggc att gca aat acc gct cgc attacc 2780 Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr Ala Arg Ile Thr680 685 690 aga gat aaa att ggc ttt gct ggt tct gat ggt gca gtt gat acaaac 2828 Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala Val Asp Thr Asn695 700 705 aaa cct tat ctt gat caa gac aag cta caa gtt ggc aat gtt aagatt 2876 Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly Asn Val Lys Ile710 715 720 acc aac act ggc att aac gca ggt ggt aaa gcc atc aca ggg ctgtcc 2924 Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile Thr Gly Leu Ser725 730 735 cca aca ctg cct agc att gcc gat caa agt agc cgc aac ata gaactg 2972 Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg Asn Ile Glu Leu740 745 750 755 ggc aat aca atc caa gac aaa gac aaa tcc aac gct gcc agcatt aat 3020 Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala Ala Ser IleAsn 760 765 770 gat ata tta aat aca ggc ttt aac cta aaa aat aat aac aacccc att 3068 Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn Asn Asn ProIle 775 780 785 gac ttt gtc tcc act tat gac att gtt gac ttt gcc aat ggcaat gcc 3116 Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala Asn Gly AsnAla 790 795 800 acc acc gcc aca gta acc cat gat acc gct aac aaa acc agtaaa gtg 3164 Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys Thr Ser LysVal 805 810 815 gta tat gat gtg aat gtg gat gat aca acc att cat cta acaggc act 3212 Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His Leu Thr GlyThr 820 825 830 835 gat gac aat aaa aaa ctt ggc gtc aaa acc acc aaa ctgaac aaa aca 3260 Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys Leu AsnLys Thr 840 845 850 agt gct aat ggt aat aca gca act aac ttt aat gtt aactct agt gat 3308 Ser Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn Val Asn SerSer Asp 855 860 865 gaa gat gcc ctt gtt aac gcc aaa gac atc gcc gaa aatcta aac acc 3356 Glu Asp Ala Leu Val Asn Ala Lys Asp Ile Ala Glu Asn LeuAsn Thr 870 875 880 cta gcc aag gaa att cac acc acc aaa ggc aca gca gacacc gcc cta 3404 Leu Ala Lys Glu Ile His Thr Thr Lys Gly Thr Ala Asp ThrAla Leu 885 890 895 caa acc ttt acc gtt aaa aag gta gat gaa aat aat aatgct gat gac 3452 Gln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn Asn AlaAsp Asp 900 905 910 915 gcc aac gcc atc acc gtg ggt caa aag aac gca aataat caa gtc aac 3500 Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn AsnGln Val Asn 920 925 930 acc cta aca ctc aaa ggt gaa aac ggt ctt aat attaaa acc gac aaa 3548 Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile LysThr Asp Lys 935 940 945 aat ggt acg gtt acc ttt ggc att aac acc aca agcggt ctt aaa gcc 3596 Asn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser GlyLeu Lys Ala 950 955 960 ggc aaa agc acc cta aac gac ggt ggc ttg tct attaaa aac ccc act 3644 Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile LysAsn Pro Thr 965 970 975 ggt agc gaa caa atc caa gtc ggt gct gat ggc gtgaag ttt gcc aag 3692 Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val LysPhe Ala Lys 980 985 990 995 gtt aat aat aat ggt gtt gta ggt gct ggc attgat ggc aca act cgc 3740 Val Asn Asn Asn Gly Val Val Gly Ala Gly Ile AspGly Thr Thr Arg 1000 1005 1010 att acc aga gat gaa att ggc ttt act gggact aat ggc tca ctt gat 3788 Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly ThrAsn Gly Ser Leu Asp 1015 1020 1025 aaa agc aaa ccc cac cta agc aaa gacggc att aac gca ggt ggt aaa 3836 Lys Ser Lys Pro His Leu Ser Lys Asp GlyIle Asn Ala Gly Gly Lys 1030 1035 1040 aag att acc aac att caa tca ggtgag att gcc caa aac agc cat gat 3884 Lys Ile Thr Asn Ile Gln Ser Gly GluIle Ala Gln Asn Ser His Asp 1045 1050 1055 gct gtg aca ggc ggc aag atttat gat tta aaa acc gaa ctt gaa aac 3932 Ala Val Thr Gly Gly Lys Ile TyrAsp Leu Lys Thr Glu Leu Glu Asn 1060 1065 1070 1075 aaa atc agc agt actgcc aaa aca gca caa aac tca tta cac gaa ttc 3980 Lys Ile Ser Ser Thr AlaLys Thr Ala Gln Asn Ser Leu His Glu Phe 1080 1085 1090 tca gta gca gatgaa caa ggt aat aac ttt acg gtt agt aac cct tac 4028 Ser Val Ala Asp GluGln Gly Asn Asn Phe Thr Val Ser Asn Pro Tyr 1095 1100 1105 tcc agt tatgac acc tca aag acc tct gat gtc atc acc ttt gca ggt 4076 Ser Ser Tyr AspThr Ser Lys Thr Ser Asp Val Ile Thr Phe Ala Gly 1110 1115 1120 gaa aacggc att acc acc aag gta aat aaa ggt gtg gtg cgt gtg ggc 4124 Glu Asn GlyIle Thr Thr Lys Val Asn Lys Gly Val Val Arg Val Gly 1125 1130 1135 attgac caa acc aaa ggc tta acc acg cct aag ctg acc gtg ggt aat 4172 Ile AspGln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr Val Gly Asn 1140 1145 11501155 aat aat ggc aaa ggc att gtc att gac agc caa aat ggt caa aat acc4220 Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn Gly Gln Asn Thr1160 1165 1170 atc aca gga cta agc aac act cta gct aat gtt acc aat gataaa ggt 4268 Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn Val Thr Asn Asp LysGly 1175 1180 1185 agc gta cgc acc aca gaa cag ggc aat ata atc aaa gacgaa gac aaa 4316 Ser Val Arg Thr Thr Glu Gln Gly Asn Ile Ile Lys Asp GluAsp Lys 1190 1195 1200 acc cgt gcc gcc agc att gtt gat gtg cta agc gcaggc ttt aac ttg 4364 Thr Arg Ala Ala Ser Ile Val Asp Val Leu Ser Ala GlyPhe Asn Leu 1205 1210 1215 caa ggc aat ggt gaa gcg gtt gac ttt gtc tccact tat gac acc gtc 4412 Gln Gly Asn Gly Glu Ala Val Asp Phe Val Ser ThrTyr Asp Thr Val 1220 1225 1230 1235 aac ttt gcc gat ggc aat gcc acc accgct aag gtg acc tat gat gac 4460 Asn Phe Ala Asp Gly Asn Ala Thr Thr AlaLys Val Thr Tyr Asp Asp 1240 1245 1250 aca agc aaa acc agt aaa gtg gtctat gat gtc aat gtg gat gat aca 4508 Thr Ser Lys Thr Ser Lys Val Val TyrAsp Val Asn Val Asp Asp Thr 1255 1260 1265 acc att gaa gtt aaa gat aaaaaa ctt ggc gta aaa acc acc aca ttg 4556 Thr Ile Glu Val Lys Asp Lys LysLeu Gly Val Lys Thr Thr Thr Leu 1270 1275 1280 acc agt act ggc aca ggtgct aat aaa ttt gcc cta agc aat caa gct 4604 Thr Ser Thr Gly Thr Gly AlaAsn Lys Phe Ala Leu Ser Asn Gln Ala 1285 1290 1295 act ggc gat gcg cttgtc aag gcc agt gat atc gtt gct cat cta aac 4652 Thr Gly Asp Ala Leu ValLys Ala Ser Asp Ile Val Ala His Leu Asn 1300 1305 1310 1315 acc tta tctggc gac atc caa act gcc aaa ggg gca agc caa gcg aac 4700 Thr Leu Ser GlyAsp Ile Gln Thr Ala Lys Gly Ala Ser Gln Ala Asn 1320 1325 1330 aac tcagca ggc tat gtg gat gct gat ggc aat aag gtc atc tat gac 4748 Asn Ser AlaGly Tyr Val Asp Ala Asp Gly Asn Lys Val Ile Tyr Asp 1335 1340 1345 agtacc gat aac aag tac tat caa gcc aaa aat gat ggc aca gtt gat 4796 Ser ThrAsp Asn Lys Tyr Tyr Gln Ala Lys Asn Asp Gly Thr Val Asp 1350 1355 1360aaa acc aaa gaa gtt gcc aaa gac aaa ctg gtc gcc caa gcc caa acc 4844 LysThr Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln Ala Gln Thr 1365 13701375 cca gat ggc aca ttg gct caa atg aat gtc aaa tca gtc att aac aaa4892 Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser Val Ile Asn Lys1380 1385 1390 1395 gaa caa gta aat gat gcc aat aaa aag caa ggc atc aatgaa gac aac 4940 Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile Asn GluAsp Asn 1400 1405 1410 gcc ttt gtt aaa gga ctt gaa aaa gcc gct tct gataac aaa acc aaa 4988 Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp AsnLys Thr Lys 1415 1420 1425 aac gcc gca gta act gtg ggt gat tta aat gccgtt gcc caa aca ccg 5036 Asn Ala Ala Val Thr Val Gly Asp Leu Asn Ala ValAla Gln Thr Pro 1430 1435 1440 ctg acc ttt gca ggg gat aca ggc aca acggct aaa aaa ctg ggc gag 5084 Leu Thr Phe Ala Gly Asp Thr Gly Thr Thr AlaLys Lys Leu Gly Glu 1445 1450 1455 act ttg acc atc aaa ggt ggg caa acagac acc aat aag cta acc gat 5132 Thr Leu Thr Ile Lys Gly Gly Gln Thr AspThr Asn Lys Leu Thr Asp 1460 1465 1470 1475 aat aac atc ggt gtg gta gcaggt act gat ggc ttc act gtc aaa ctt 5180 Asn Asn Ile Gly Val Val Ala GlyThr Asp Gly Phe Thr Val Lys Leu 1480 1485 1490 gcc aaa gac cta acc aatctt aac agc gtt aat gca ggt ggc acc aaa 5228 Ala Lys Asp Leu Thr Asn LeuAsn Ser Val Asn Ala Gly Gly Thr Lys 1495 1500 1505 att gat gac aaa ggcgtg tct ttt gta gac tca agc ggt caa gcc aaa 5276 Ile Asp Asp Lys Gly ValSer Phe Val Asp Ser Ser Gly Gln Ala Lys 1510 1515 1520 gca aac acc cctgtg cta agt gcc aat ggg ctg gac ctg ggt ggc aag 5324 Ala Asn Thr Pro ValLeu Ser Ala Asn Gly Leu Asp Leu Gly Gly Lys 1525 1530 1535 gtc atc agtaat gtg ggc aaa ggc aca aaa gat acc gac gct gcc aat 5372 Val Ile Ser AsnVal Gly Lys Gly Thr Lys Asp Thr Asp Ala Ala Asn 1540 1545 1550 1555 gtacaa cag tta aac gaa gta cgc aac ttg ttg ggt ctt ggt aat gct 5420 Val GlnGln Leu Asn Glu Val Arg Asn Leu Leu Gly Leu Gly Asn Ala 1560 1565 1570ggt aat gat aac gct gac ggc aat cag gta aac att gcc gac atc aaa 5468 GlyAsn Asp Asn Ala Asp Gly Asn Gln Val Asn Ile Ala Asp Ile Lys 1575 15801585 aaa gac cca aat tca ggt tca tca tct aac cgc act gtc atc aaa gca5516 Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn Arg Thr Val Ile Lys Ala1590 1595 1600 ggc acg gta ctt ggc ggt aaa ggt aat aac gat acc gaa aaactt gcc 5564 Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp Thr Glu Lys LeuAla 1605 1610 1615 act ggt ggt ata caa gtg ggc gtg gat aaa gac ggc aacgct aac ggc 5612 Thr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly Asn AlaAsn Gly 1620 1625 1630 1635 gat tta agc aat gtt tgg gtc aaa acc caa aaagat ggc agc aaa aaa 5660 Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys AspGly Ser Lys Lys 1640 1645 1650 gcc ctg ctc gcc act tat aac gcc gca ggtcag acc aac tat ttg acc 5708 Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly GlnThr Asn Tyr Leu Thr 1655 1660 1665 aac aac ccc gca gaa gcc att gac agaata aat gaa caa ggt atc cgc 5756 Asn Asn Pro Ala Glu Ala Ile Asp Arg IleAsn Glu Gln Gly Ile Arg 1670 1675 1680 ttc ttc cat gtc aac gat ggc aatcaa gag cct gtg gta caa ggg cgt 5804 Phe Phe His Val Asn Asp Gly Asn GlnGlu Pro Val Val Gln Gly Arg 1685 1690 1695 aac ggc att gac tca agt gcctca ggc aag cac tca gtg gcg ata ggt 5852 Asn Gly Ile Asp Ser Ser Ala SerGly Lys His Ser Val Ala Ile Gly 1700 1705 1710 1715 ttc cag gcc aag gcagat ggt gaa gcc gcc gtt gcc ata ggc aga caa 5900 Phe Gln Ala Lys Ala AspGly Glu Ala Ala Val Ala Ile Gly Arg Gln 1720 1725 1730 acc caa gca ggcaac caa tcc atc gcc atc ggt gat aac gca caa gcc 5948 Thr Gln Ala Gly AsnGln Ser Ile Ala Ile Gly Asp Asn Ala Gln Ala 1735 1740 1745 acg ggc gatcaa tcc atc gcc atc ggt aca ggc aat gtg gta gca ggt 5996 Thr Gly Asp GlnSer Ile Ala Ile Gly Thr Gly Asn Val Val Ala Gly 1750 1755 1760 aag cactct ggt gcc atc ggc gac cca agc act gtt aag gct gat aac 6044 Lys His SerGly Ala Ile Gly Asp Pro Ser Thr Val Lys Ala Asp Asn 1765 1770 1775 agttac agt gtg ggt aat aac aac cag ttt acc gat gcc act caa acc 6092 Ser TyrSer Val Gly Asn Asn Asn Gln Phe Thr Asp Ala Thr Gln Thr 1780 1785 17901795 gat gtc ttt ggt gtg ggc aat aac atc acc gtg acc gaa agt aac tcg6140 Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val Thr Glu Ser Asn Ser1800 1805 1810 gtt gcc tta ggt tca aac tct gcc atc agt gca ggc aca cacgca ggc 6188 Val Ala Leu Gly Ser Asn Ser Ala Ile Ser Ala Gly Thr His AlaGly 1815 1820 1825 aca caa gcc aaa aaa tct gac ggc aca gca ggt aca accacc aca gca 6236 Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala Gly Thr Thr ThrThr Ala 1830 1835 1840 ggt gca acc ggt acg gtt aaa ggc ttt gct gga caaacg gcg gtt ggt 6284 Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly Gln ThrAla Val Gly 1845 1850 1855 gcg gtc tcc gtg ggt gcc tca ggt gct gaa cgccgt atc caa aat gtg 6332 Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg ArgIle Gln Asn Val 1860 1865 1870 1875 gca gca ggt gag gtc agt gcc acc agcacc gat gcg gtc aat ggt agc 6380 Ala Ala Gly Glu Val Ser Ala Thr Ser ThrAsp Ala Val Asn Gly Ser 1880 1885 1890 cag ttg tac aaa gcc acc caa agcatt gcc aac gca acc aat gag ctt 6428 Gln Leu Tyr Lys Ala Thr Gln Ser IleAla Asn Ala Thr Asn Glu Leu 1895 1900 1905 gac cat cgt atc cac caa aacgaa aat aag gcc aat gca ggg att tca 6476 Asp His Arg Ile His Gln Asn GluAsn Lys Ala Asn Ala Gly Ile Ser 1910 1915 1920 tca gcg atg gcg atg gcgtcc atg cca caa gcc tac att cct ggc aga 6524 Ser Ala Met Ala Met Ala SerMet Pro Gln Ala Tyr Ile Pro Gly Arg 1925 1930 1935 tcc atg gtt acc gggggt att gcc acc cac aac ggt caa ggt gcg gtg 6572 Ser Met Val Thr Gly GlyIle Ala Thr His Asn Gly Gln Gly Ala Val 1940 1945 1950 1955 gca gtg ggactg tcg aag ctg tcg gat aat ggt caa tgg gta ttt aaa 6620 Ala Val Gly LeuSer Lys Leu Ser Asp Asn Gly Gln Trp Val Phe Lys 1960 1965 1970 atc aatggt tca gcc gat acc caa ggc cat gta ggg gcg gca gtt ggt 6668 Ile Asn GlySer Ala Asp Thr Gln Gly His Val Gly Ala Ala Val Gly 1975 1980 1985 gcaggt ttt cac ttt taagccataa atcgcaagat tttacttaaa aatcaatctc 6723 Ala GlyPhe His Phe 1990 accatagttg tataaaacag catcagcatc agtcatatta ctgatgctgatgttttttat 6783 cacttaaacc attttaccgc tcaagtgatt ctctttcacc atgaccaaatcgccattgat 6843 cataggtaaa cttattgagt aaattttatc aatgtagttg ttagatatggttaaaattgt 6903 gccattgacc aaaaaatgac cgatttatcc cgaaaatttc tgattatgatccgttgacct 6963 gcaggtcgac 6973 2 5976 DNA Moraxella catarrhalis 2gtgatcggtg caacgctcag tggcagtgct tatgctcaaa aaaaagatac caaacatatc 60gcaattggtg aacaaaacca gccaagacgc tcaggcactg ccaaggcgga cggtgatcga 120gccattgcta ttggtgaaaa tgctaacgca cagggcggtc aagccatcgc catcggtagt 180agtaataaaa ctgtcaatgg aagcagtttg gataagatag gtaccgatgc tacgggtcaa 240gagtccatcg ccatcggtgg tgatgtaaag gctagtggtg atgcctcgat tgccatcggt 300agtgatgact tacatttgct tgatcagcat ggtaatccta aacatccgaa aggtactctg 360attaacgatc ttattaacgg ccatgcagta ttaaaagaaa tacgaagctc aaaggataat 420gatgtaaaat atagacgcac aaccgcaagc ggacacgcca gtactgcagt gggagccatg 480tcatatgcac agggtcattt ttccaacgcc tttggtacac gggcaacagc taaaagtgcc 540tattccttgg cagtgggtct tgccgccaca gccgagggcc aatctacaat cgctattggt 600tctgatgcaa catctagctc gttgggagcg atagcccttg gtgcaggtac tcgtgctcag 660ctacagggca gtattgccct aggtcaaggt tctgttgtca ctcagagtga taataattct 720agaccggcct atacaccaaa tacccaggca ctagacccca agtttcaagc caccaataat 780acgaaggcgg gtccactttc cattggtagt aactctatca aacgtaaaat catcaatgtc 840ggtgcaggtg ttaataaaac cgatgcggtc aatgtggcac agctagaagc ggtggtgaag 900tgggctaagg agcgtagaat tacttttcag ggtgatgata acagtactga cgtaaaaata 960ggtttggata atactttaac tattaaaggt ggtgcagaga ccaacgcatt aaccgataat 1020aatatcggtg tggtaaaaga ggctgataat agtggtctga aagttaaact tgctaaaact 1080ttaaacaatc ttactgaggt gaatacaact acattaaatg ccacaaccac agttaaggta 1140ggtagtagta gtagtactac agctgaatta ttgagtgata gtttaacctt tacccagccc 1200aatacaggca gtcaaagcac aagcaaaacc gtctatggcg ttaatggggt gaagtttact 1260aataatgcag aaacaacagc agcaatcggc actactcgta ttaccagaga taaaattggc 1320tttgctcgag atggtgatgt tgatgaaaaa caagcaccat atttggataa aaaacaactt 1380aaagtgggta gtgttgcaat taccatagac aatggcattg atgcaggtaa taaaaagatc 1440agtaatcttg ccaaaggtag cagtgctaac gatgcggtta ccatcgaaca gctcaaagcc 1500gccaagccta ctttaaacgc aggcgctggc atcagtgtca cacctactga aatatcagtt 1560gatgctaaga gtggcaatgt taccgcccca acttacaaca ttggcgtgaa aaccaccgag 1620cttaacagtg atggcactag tgataaattt agtgttaagg gtagtggtac gaacaatagc 1680ttagttaccg ccgaacattt ggcaagctat ctaaatgaag tcaatcgaac ggctgacagt 1740gctctacaaa gctttaccgt taaagaagaa gacgatgatg acgccaacgc tatcaccgtg 1800gctaaagata cgacaaaaaa tgccggcgca gtcagcatct taaaactcaa aggtaaaaac 1860ggtctaacgg ttgctaccaa aaaagatggt acggttacct ttgggcttag ccaagatagc 1920ggtctgacca ttggcaaaag caccctaaac aacgatggct tgactgttaa agataccaac 1980gaacaaatcc aagtcggtgc taatggcatt aaatttacta atgtgaatgg tagtaatcca 2040ggtactggca ttgcaaatac cgctcgcatt accagagata aaattggctt tgctggttct 2100gatggtgcag ttgatacaaa caaaccttat cttgatcaag acaagctaca agttggcaat 2160gttaagatta ccaacactgg cattaacgca ggtggtaaag ccatcacagg gctgtcccca 2220acactgccta gcattgccga tcaaagtagc cgcaacatag aactgggcaa tacaatccaa 2280gacaaagaca aatccaacgc tgccagcatt aatgatatat taaatacagg ctttaaccta 2340aaaaataata acaaccccat tgactttgtc tccacttatg acattgttga ctttgccaat 2400ggcaatgcca ccaccgccac agtaacccat gataccgcta acaaaaccag taaagtggta 2460tatgatgtga atgtggatga tacaaccatt catctaacag gcactgatga caataaaaaa 2520cttggcgtca aaaccaccaa actgaacaaa acaagtgcta atggtaatac agcaactaac 2580tttaatgtta actctagtga tgaagatgcc cttgttaacg ccaaagacat cgccgaaaat 2640ctaaacaccc tagccaagga aattcacacc accaaaggca cagcagacac cgccctacaa 2700acctttaccg ttaaaaaggt agatgaaaat aataatgctg atgacgccaa cgccatcacc 2760gtgggtcaaa agaacgcaaa taatcaagtc aacaccctaa cactcaaagg tgaaaacggt 2820cttaatatta aaaccgacaa aaatggtacg gttacctttg gcattaacac cacaagcggt 2880cttaaagccg gcaaaagcac cctaaacgac ggtggcttgt ctattaaaaa ccccactggt 2940agcgaacaaa tccaagtcgg tgctgatggc gtgaagtttg ccaaggttaa taataatggt 3000gttgtaggtg ctggcattga tggcacaact cgcattacca gagatgaaat tggctttact 3060gggactaatg gctcacttga taaaagcaaa ccccacctaa gcaaagacgg cattaacgca 3120ggtggtaaaa agattaccaa cattcaatca ggtgagattg cccaaaacag ccatgatgct 3180gtgacaggcg gcaagattta tgatttaaaa accgaacttg aaaacaaaat cagcagtact 3240gccaaaacag cacaaaactc attacacgaa ttctcagtag cagatgaaca aggtaataac 3300tttacggtta gtaaccctta ctccagttat gacacctcaa agacctctga tgtcatcacc 3360tttgcaggtg aaaacggcat taccaccaag gtaaataaag gtgtggtgcg tgtgggcatt 3420gaccaaacca aaggcttaac cacgcctaag ctgaccgtgg gtaataataa tggcaaaggc 3480attgtcattg acagccaaaa tggtcaaaat accatcacag gactaagcaa cactctagct 3540aatgttacca atgataaagg tagcgtacgc accacagaac agggcaatat aatcaaagac 3600gaagacaaaa cccgtgccgc cagcattgtt gatgtgctaa gcgcaggctt taacttgcaa 3660ggcaatggtg aagcggttga ctttgtctcc acttatgaca ccgtcaactt tgccgatggc 3720aatgccacca ccgctaaggt gacctatgat gacacaagca aaaccagtaa agtggtctat 3780gatgtcaatg tggatgatac aaccattgaa gttaaagata aaaaacttgg cgtaaaaacc 3840accacattga ccagtactgg cacaggtgct aataaatttg ccctaagcaa tcaagctact 3900ggcgatgcgc ttgtcaaggc cagtgatatc gttgctcatc taaacacctt atctggcgac 3960atccaaactg ccaaaggggc aagccaagcg aacaactcag caggctatgt ggatgctgat 4020ggcaataagg tcatctatga cagtaccgat aacaagtact atcaagccaa aaatgatggc 4080acagttgata aaaccaaaga agttgccaaa gacaaactgg tcgcccaagc ccaaacccca 4140gatggcacat tggctcaaat gaatgtcaaa tcagtcatta acaaagaaca agtaaatgat 4200gccaataaaa agcaaggcat caatgaagac aacgcctttg ttaaaggact tgaaaaagcc 4260gcttctgata acaaaaccaa aaacgccgca gtaactgtgg gtgatttaaa tgccgttgcc 4320caaacaccgc tgacctttgc aggggataca ggcacaacgg ctaaaaaact gggcgagact 4380ttgaccatca aaggtgggca aacagacacc aataagctaa ccgataataa catcggtgtg 4440gtagcaggta ctgatggctt cactgtcaaa cttgccaaag acctaaccaa tcttaacagc 4500gttaatgcag gtggcaccaa aattgatgac aaaggcgtgt cttttgtaga ctcaagcggt 4560caagccaaag caaacacccc tgtgctaagt gccaatgggc tggacctggg tggcaaggtc 4620atcagtaatg tgggcaaagg cacaaaagat accgacgctg ccaatgtaca acagttaaac 4680gaagtacgca acttgttggg tcttggtaat gctggtaatg ataacgctga cggcaatcag 4740gtaaacattg ccgacatcaa aaaagaccca aattcaggtt catcatctaa ccgcactgtc 4800atcaaagcag gcacggtact tggcggtaaa ggtaataacg ataccgaaaa acttgccact 4860ggtggtatac aagtgggcgt ggataaagac ggcaacgcta acggcgattt aagcaatgtt 4920tgggtcaaaa cccaaaaaga tggcagcaaa aaagccctgc tcgccactta taacgccgca 4980ggtcagacca actatttgac caacaacccc gcagaagcca ttgacagaat aaatgaacaa 5040ggtatccgct tcttccatgt caacgatggc aatcaagagc ctgtggtaca agggcgtaac 5100ggcattgact caagtgcctc aggcaagcac tcagtggcga taggtttcca ggccaaggca 5160gatggtgaag ccgccgttgc cataggcaga caaacccaag caggcaacca atccatcgcc 5220atcggtgata acgcacaagc cacgggcgat caatccatcg ccatcggtac aggcaatgtg 5280gtagcaggta agcactctgg tgccatcggc gacccaagca ctgttaaggc tgataacagt 5340tacagtgtgg gtaataacaa ccagtttacc gatgccactc aaaccgatgt ctttggtgtg 5400ggcaataaca tcaccgtgac cgaaagtaac tcggttgcct taggttcaaa ctctgccatc 5460agtgcaggca cacacgcagg cacacaagcc aaaaaatctg acggcacagc aggtacaacc 5520accacagcag gtgcaaccgg tacggttaaa ggctttgctg gacaaacggc ggttggtgcg 5580gtctccgtgg gtgcctcagg tgctgaacgc cgtatccaaa atgtggcagc aggtgaggtc 5640agtgccacca gcaccgatgc ggtcaatggt agccagttgt acaaagccac ccaaagcatt 5700gccaacgcaa ccaatgagct tgaccatcgt atccaccaaa acgaaaataa ggccaatgca 5760gggatttcat cagcgatggc gatggcgtcc atgccacaag cctacattcc tggcagatcc 5820atggttaccg ggggtattgc cacccacaac ggtcaaggtg cggtggcagt gggactgtcg 5880aagctgtcgg ataatggtca atgggtattt aaaatcaatg gttcagccga tacccaaggc 5940catgtagggg cggcagttgg tgcaggtttt cacttt 5976 3 1992 PRT Moraxellacatarrhalis 3 Met Ile Gly Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln LysLys Asp 1 5 10 15 Thr Lys His Ile Ala Ile Gly Glu Gln Asn Gln Pro ArgArg Ser Gly 20 25 30 Thr Ala Lys Ala Asp Gly Asp Arg Ala Ile Ala Ile GlyGlu Asn Ala 35 40 45 Asn Ala Gln Gly Gly Gln Ala Ile Ala Ile Gly Ser SerAsn Lys Thr 50 55 60 Val Asn Gly Ser Ser Leu Asp Lys Ile Gly Thr Asp AlaThr Gly Gln 65 70 75 80 Glu Ser Ile Ala Ile Gly Gly Asp Val Lys Ala SerGly Asp Ala Ser 85 90 95 Ile Ala Ile Gly Ser Asp Asp Leu His Leu Leu AspGln His Gly Asn 100 105 110 Pro Lys His Pro Lys Gly Thr Leu Ile Asn AspLeu Ile Asn Gly His 115 120 125 Ala Val Leu Lys Glu Ile Arg Ser Ser LysAsp Asn Asp Val Lys Tyr 130 135 140 Arg Arg Thr Thr Ala Ser Gly His AlaSer Thr Ala Val Gly Ala Met 145 150 155 160 Ser Tyr Ala Gln Gly His PheSer Asn Ala Phe Gly Thr Arg Ala Thr 165 170 175 Ala Lys Ser Ala Tyr SerLeu Ala Val Gly Leu Ala Ala Thr Ala Glu 180 185 190 Gly Gln Ser Thr IleAla Ile Gly Ser Asp Ala Thr Ser Ser Ser Leu 195 200 205 Gly Ala Ile AlaLeu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly Ser 210 215 220 Ile Ala LeuGly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn Ser 225 230 235 240 ArgPro Ala Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe Gln 245 250 255Ala Thr Asn Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn Ser 260 265270 Ile Lys Arg Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr Asp 275280 285 Ala Val Asn Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys Glu290 295 300 Arg Arg Ile Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val LysIle 305 310 315 320 Gly Leu Asp Asn Thr Leu Thr Ile Lys Gly Gly Ala GluThr Asn Ala 325 330 335 Leu Thr Asp Asn Asn Ile Gly Val Val Lys Glu AlaAsp Asn Ser Gly 340 345 350 Leu Lys Val Lys Leu Ala Lys Thr Leu Asn AsnLeu Thr Glu Val Asn 355 360 365 Thr Thr Thr Leu Asn Ala Thr Thr Thr ValLys Val Gly Ser Ser Ser 370 375 380 Ser Thr Thr Ala Glu Leu Leu Ser AspSer Leu Thr Phe Thr Gln Pro 385 390 395 400 Asn Thr Gly Ser Gln Ser ThrSer Lys Thr Val Tyr Gly Val Asn Gly 405 410 415 Val Lys Phe Thr Asn AsnAla Glu Thr Thr Ala Ala Ile Gly Thr Thr 420 425 430 Arg Ile Thr Arg AspLys Ile Gly Phe Ala Arg Asp Gly Asp Val Asp 435 440 445 Glu Lys Gln AlaPro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly Ser 450 455 460 Val Ala IleThr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys Ile 465 470 475 480 SerAsn Leu Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile Glu 485 490 495Gln Leu Lys Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile Ser 500 505510 Val Thr Pro Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val Thr 515520 525 Ala Pro Thr Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser Asp530 535 540 Gly Thr Ser Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn AsnSer 545 550 555 560 Leu Val Thr Ala Glu His Leu Ala Ser Tyr Leu Asn GluVal Asn Arg 565 570 575 Thr Ala Asp Ser Ala Leu Gln Ser Phe Thr Val LysGlu Glu Asp Asp 580 585 590 Asp Asp Ala Asn Ala Ile Thr Val Ala Lys AspThr Thr Lys Asn Ala 595 600 605 Gly Ala Val Ser Ile Leu Lys Leu Lys GlyLys Asn Gly Leu Thr Val 610 615 620 Ala Thr Lys Lys Asp Gly Thr Val ThrPhe Gly Leu Ser Gln Asp Ser 625 630 635 640 Gly Leu Thr Ile Gly Lys SerThr Leu Asn Asn Asp Gly Leu Thr Val 645 650 655 Lys Asp Thr Asn Glu GlnIle Gln Val Gly Ala Asn Gly Ile Lys Phe 660 665 670 Thr Asn Val Asn GlySer Asn Pro Gly Thr Gly Ile Ala Asn Thr Ala 675 680 685 Arg Ile Thr ArgAsp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala Val 690 695 700 Asp Thr AsnLys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly Asn 705 710 715 720 ValLys Ile Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile Thr 725 730 735Gly Leu Ser Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg Asn 740 745750 Ile Glu Leu Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala Ala 755760 765 Ser Ile Asn Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn Asn770 775 780 Asn Pro Ile Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe AlaAsn 785 790 795 800 Gly Asn Ala Thr Thr Ala Thr Val Thr His Asp Thr AlaAsn Lys Thr 805 810 815 Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp ThrThr Ile His Leu 820 825 830 Thr Gly Thr Asp Asp Asn Lys Lys Leu Gly ValLys Thr Thr Lys Leu 835 840 845 Asn Lys Thr Ser Ala Asn Gly Asn Thr AlaThr Asn Phe Asn Val Asn 850 855 860 Ser Ser Asp Glu Asp Ala Leu Val AsnAla Lys Asp Ile Ala Glu Asn 865 870 875 880 Leu Asn Thr Leu Ala Lys GluIle His Thr Thr Lys Gly Thr Ala Asp 885 890 895 Thr Ala Leu Gln Thr PheThr Val Lys Lys Val Asp Glu Asn Asn Asn 900 905 910 Ala Asp Asp Ala AsnAla Ile Thr Val Gly Gln Lys Asn Ala Asn Asn 915 920 925 Gln Val Asn ThrLeu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile Lys 930 935 940 Thr Asp LysAsn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser Gly 945 950 955 960 LeuLys Ala Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile Lys 965 970 975Asn Pro Thr Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val Lys 980 985990 Phe Ala Lys Val Asn Asn Asn Gly Val Val Gly Ala Gly Ile Asp Gly 9951000 1005 Thr Thr Arg Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly Thr AsnGly 1010 1015 1020 Ser Leu Asp Lys Ser Lys Pro His Leu Ser Lys Asp GlyIle Asn Ala 1025 1030 1035 1040 Gly Gly Lys Lys Ile Thr Asn Ile Gln SerGly Glu Ile Ala Gln Asn 1045 1050 1055 Ser His Asp Ala Val Thr Gly GlyLys Ile Tyr Asp Leu Lys Thr Glu 1060 1065 1070 Leu Glu Asn Lys Ile SerSer Thr Ala Lys Thr Ala Gln Asn Ser Leu 1075 1080 1085 His Glu Phe SerVal Ala Asp Glu Gln Gly Asn Asn Phe Thr Val Ser 1090 1095 1100 Asn ProTyr Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile Thr 1105 1110 11151120 Phe Ala Gly Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val Val1125 1130 1135 Arg Val Gly Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro LysLeu Thr 1140 1145 1150 Val Gly Asn Asn Asn Gly Lys Gly Ile Val Ile AspSer Gln Asn Gly 1155 1160 1165 Gln Asn Thr Ile Thr Gly Leu Ser Asn ThrLeu Ala Asn Val Thr Asn 1170 1175 1180 Asp Lys Gly Ser Val Arg Thr ThrGlu Gln Gly Asn Ile Ile Lys Asp 1185 1190 1195 1200 Glu Asp Lys Thr ArgAla Ala Ser Ile Val Asp Val Leu Ser Ala Gly 1205 1210 1215 Phe Asn LeuGln Gly Asn Gly Glu Ala Val Asp Phe Val Ser Thr Tyr 1220 1225 1230 AspThr Val Asn Phe Ala Asp Gly Asn Ala Thr Thr Ala Lys Val Thr 1235 12401245 Tyr Asp Asp Thr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn Val1250 1255 1260 Asp Asp Thr Thr Ile Glu Val Lys Asp Lys Lys Leu Gly ValLys Thr 1265 1270 1275 1280 Thr Thr Leu Thr Ser Thr Gly Thr Gly Ala AsnLys Phe Ala Leu Ser 1285 1290 1295 Asn Gln Ala Thr Gly Asp Ala Leu ValLys Ala Ser Asp Ile Val Ala 1300 1305 1310 His Leu Asn Thr Leu Ser GlyAsp Ile Gln Thr Ala Lys Gly Ala Ser 1315 1320 1325 Gln Ala Asn Asn SerAla Gly Tyr Val Asp Ala Asp Gly Asn Lys Val 1330 1335 1340 Ile Tyr AspSer Thr Asp Asn Lys Tyr Tyr Gln Ala Lys Asn Asp Gly 1345 1350 1355 1360Thr Val Asp Lys Thr Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln 13651370 1375 Ala Gln Thr Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys SerVal 1380 1385 1390 Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys Lys GlnGly Ile Asn 1395 1400 1405 Glu Asp Asn Ala Phe Val Lys Gly Leu Glu LysAla Ala Ser Asp Asn 1410 1415 1420 Lys Thr Lys Asn Ala Ala Val Thr ValGly Asp Leu Asn Ala Val Ala 1425 1430 1435 1440 Gln Thr Pro Leu Thr PheAla Gly Asp Thr Gly Thr Thr Ala Lys Lys 1445 1450 1455 Leu Gly Glu ThrLeu Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn Lys 1460 1465 1470 Leu ThrAsp Asn Asn Ile Gly Val Val Ala Gly Thr Asp Gly Phe Thr 1475 1480 1485Val Lys Leu Ala Lys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly 14901495 1500 Gly Thr Lys Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser SerGly 1505 1510 1515 1520 Gln Ala Lys Ala Asn Thr Pro Val Leu Ser Ala AsnGly Leu Asp Leu 1525 1530 1535 Gly Gly Lys Val Ile Ser Asn Val Gly LysGly Thr Lys Asp Thr Asp 1540 1545 1550 Ala Ala Asn Val Gln Gln Leu AsnGlu Val Arg Asn Leu Leu Gly Leu 1555 1560 1565 Gly Asn Ala Gly Asn AspAsn Ala Asp Gly Asn Gln Val Asn Ile Ala 1570 1575 1580 Asp Ile Lys LysAsp Pro Asn Ser Gly Ser Ser Ser Asn Arg Thr Val 1585 1590 1595 1600 IleLys Ala Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp Thr Glu 1605 16101615 Lys Leu Ala Thr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly Asn1620 1625 1630 Ala Asn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln LysAsp Gly 1635 1640 1645 Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala AlaGly Gln Thr Asn 1650 1655 1660 Tyr Leu Thr Asn Asn Pro Ala Glu Ala IleAsp Arg Ile Asn Glu Gln 1665 1670 1675 1680 Gly Ile Arg Phe Phe His ValAsn Asp Gly Asn Gln Glu Pro Val Val 1685 1690 1695 Gln Gly Arg Asn GlyIle Asp Ser Ser Ala Ser Gly Lys His Ser Val 1700 1705 1710 Ala Ile GlyPhe Gln Ala Lys Ala Asp Gly Glu Ala Ala Val Ala Ile 1715 1720 1725 GlyArg Gln Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly Asp Asn 1730 17351740 Ala Gln Ala Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn Val1745 1750 1755 1760 Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro SerThr Val Lys 1765 1770 1775 Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn AsnGln Phe Thr Asp Ala 1780 1785 1790 Thr Gln Thr Asp Val Phe Gly Val GlyAsn Asn Ile Thr Val Thr Glu 1795 1800 1805 Ser Asn Ser Val Ala Leu GlySer Asn Ser Ala Ile Ser Ala Gly Thr 1810 1815 1820 His Ala Gly Thr GlnAla Lys Lys Ser Asp Gly Thr Ala Gly Thr Thr 1825 1830 1835 1840 Thr ThrAla Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly Gln Thr 1845 1850 1855Ala Val Gly Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg Arg Ile 18601865 1870 Gln Asn Val Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp AlaVal 1875 1880 1885 Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile AlaAsn Ala Thr 1890 1895 1900 Asn Glu Leu Asp His Arg Ile His Gln Asn GluAsn Lys Ala Asn Ala 1905 1910 1915 1920 Gly Ile Ser Ser Ala Met Ala MetAla Ser Met Pro Gln Ala Tyr Ile 1925 1930 1935 Pro Gly Arg Ser Met ValThr Gly Gly Ile Ala Thr His Asn Gly Gln 1940 1945 1950 Gly Ala Val AlaVal Gly Leu Ser Lys Leu Ser Asp Asn Gly Gln Trp 1955 1960 1965 Val PheLys Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val Gly Ala 1970 1975 1980Ala Val Gly Ala Gly Phe His Phe 1985 1990 4 1833 PRT Moraxellacatarrhalis 4 Met Ser Tyr Ala Gln Gly His Phe Ser Asn Ala Phe Gly ThrArg Ala 1 5 10 15 Thr Ala Lys Ser Ala Tyr Ser Leu Ala Val Gly Leu AlaAla Thr Ala 20 25 30 Glu Gly Gln Ser Thr Ile Ala Ile Gly Ser Asp Ala ThrSer Ser Ser 35 40 45 Leu Gly Ala Ile Ala Leu Gly Ala Gly Thr Arg Ala GlnLeu Gln Gly 50 55 60 Ser Ile Ala Leu Gly Gln Gly Ser Val Val Thr Gln SerAsp Asn Asn 65 70 75 80 Ser Arg Pro Ala Tyr Thr Pro Asn Thr Gln Ala LeuAsp Pro Lys Phe 85 90 95 Gln Ala Thr Asn Asn Thr Lys Ala Gly Pro Leu SerIle Gly Ser Asn 100 105 110 Ser Ile Lys Arg Lys Ile Ile Asn Val Gly AlaGly Val Asn Lys Thr 115 120 125 Asp Ala Val Asn Val Ala Gln Leu Glu AlaVal Val Lys Trp Ala Lys 130 135 140 Glu Arg Arg Ile Thr Phe Gln Gly AspAsp Asn Ser Thr Asp Val Lys 145 150 155 160 Ile Gly Leu Asp Asn Thr LeuThr Ile Lys Gly Gly Ala Glu Thr Asn 165 170 175 Ala Leu Thr Asp Asn AsnIle Gly Val Val Lys Glu Ala Asp Asn Ser 180 185 190 Gly Leu Lys Val LysLeu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val 195 200 205 Asn Thr Thr ThrLeu Asn Ala Thr Thr Thr Val Lys Val Gly Ser Ser 210 215 220 Ser Ser ThrThr Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe Thr Gln 225 230 235 240 ProAsn Thr Gly Ser Gln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn 245 250 255Gly Val Lys Phe Thr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr 260 265270 Thr Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val 275280 285 Asp Glu Lys Gln Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly290 295 300 Ser Val Ala Ile Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn LysLys 305 310 315 320 Ile Ser Asn Leu Ala Lys Gly Ser Ser Ala Asn Asp AlaVal Thr Ile 325 330 335 Glu Gln Leu Lys Ala Ala Lys Pro Thr Leu Asn AlaGly Ala Gly Ile 340 345 350 Ser Val Thr Pro Thr Glu Ile Ser Val Asp AlaLys Ser Gly Asn Val 355 360 365 Thr Ala Pro Thr Tyr Asn Ile Gly Val LysThr Thr Glu Leu Asn Ser 370 375 380 Asp Gly Thr Ser Asp Lys Phe Ser ValLys Gly Ser Gly Thr Asn Asn 385 390 395 400 Ser Leu Val Thr Ala Glu HisLeu Ala Ser Tyr Leu Asn Glu Val Asn 405 410 415 Arg Thr Ala Asp Ser AlaLeu Gln Ser Phe Thr Val Lys Glu Glu Asp 420 425 430 Asp Asp Asp Ala AsnAla Ile Thr Val Ala Lys Asp Thr Thr Lys Asn 435 440 445 Ala Gly Ala ValSer Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr 450 455 460 Val Ala ThrLys Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp 465 470 475 480 SerGly Leu Thr Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr 485 490 495Val Lys Asp Thr Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys 500 505510 Phe Thr Asn Val Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr 515520 525 Ala Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala530 535 540 Val Asp Thr Asn Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln ValGly 545 550 555 560 Asn Val Lys Ile Thr Asn Thr Gly Ile Asn Ala Gly GlyLys Ala Ile 565 570 575 Thr Gly Leu Ser Pro Thr Leu Pro Ser Ile Ala AspGln Ser Ser Arg 580 585 590 Asn Ile Glu Leu Gly Asn Thr Ile Gln Asp LysAsp Lys Ser Asn Ala 595 600 605 Ala Ser Ile Asn Asp Ile Leu Asn Thr GlyPhe Asn Leu Lys Asn Asn 610 615 620 Asn Asn Pro Ile Asp Phe Val Ser ThrTyr Asp Ile Val Asp Phe Ala 625 630 635 640 Asn Gly Asn Ala Thr Thr AlaThr Val Thr His Asp Thr Ala Asn Lys 645 650 655 Thr Ser Lys Val Val TyrAsp Val Asn Val Asp Asp Thr Thr Ile His 660 665 670 Leu Thr Gly Thr AspAsp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys 675 680 685 Leu Asn Lys ThrSer Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn Val 690 695 700 Asn Ser SerAsp Glu Asp Ala Leu Val Asn Ala Lys Asp Ile Ala Glu 705 710 715 720 AsnLeu Asn Thr Leu Ala Lys Glu Ile His Thr Thr Lys Gly Thr Ala 725 730 735Asp Thr Ala Leu Gln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn 740 745750 Asn Ala Asp Asp Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn 755760 765 Asn Gln Val Asn Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile770 775 780 Lys Thr Asp Lys Asn Gly Thr Val Thr Phe Gly Ile Asn Thr ThrSer 785 790 795 800 Gly Leu Lys Ala Gly Lys Ser Thr Leu Asn Asp Gly GlyLeu Ser Ile 805 810 815 Lys Asn Pro Thr Gly Ser Glu Gln Ile Gln Val GlyAla Asp Gly Val 820 825 830 Lys Phe Ala Lys Val Asn Asn Asn Gly Val ValGly Ala Gly Ile Asp 835 840 845 Gly Thr Thr Arg Ile Thr Arg Asp Glu IleGly Phe Thr Gly Thr Asn 850 855 860 Gly Ser Leu Asp Lys Ser Lys Pro HisLeu Ser Lys Asp Gly Ile Asn 865 870 875 880 Ala Gly Gly Lys Lys Ile ThrAsn Ile Gln Ser Gly Glu Ile Ala Gln 885 890 895 Asn Ser His Asp Ala ValThr Gly Gly Lys Ile Tyr Asp Leu Lys Thr 900 905 910 Glu Leu Glu Asn LysIle Ser Ser Thr Ala Lys Thr Ala Gln Asn Ser 915 920 925 Leu His Glu PheSer Val Ala Asp Glu Gln Gly Asn Asn Phe Thr Val 930 935 940 Ser Asn ProTyr Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile 945 950 955 960 ThrPhe Ala Gly Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val 965 970 975Val Arg Val Gly Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu 980 985990 Thr Val Gly Asn Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn 9951000 1005 Gly Gln Asn Thr Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn ValThr 1010 1015 1020 Asn Asp Lys Gly Ser Val Arg Thr Thr Glu Gln Gly AsnIle Ile Lys 1025 1030 1035 1040 Asp Glu Asp Lys Thr Arg Ala Ala Ser IleVal Asp Val Leu Ser Ala 1045 1050 1055 Gly Phe Asn Leu Gln Gly Asn GlyGlu Ala Val Asp Phe Val Ser Thr 1060 1065 1070 Tyr Asp Thr Val Asn PheAla Asp Gly Asn Ala Thr Thr Ala Lys Val 1075 1080 1085 Thr Tyr Asp AspThr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn 1090 1095 1100 Val AspAsp Thr Thr Ile Glu Val Lys Asp Lys Lys Leu Gly Val Lys 1105 1110 11151120 Thr Thr Thr Leu Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu1125 1130 1135 Ser Asn Gln Ala Thr Gly Asp Ala Leu Val Lys Ala Ser AspIle Val 1140 1145 1150 Ala His Leu Asn Thr Leu Ser Gly Asp Ile Gln ThrAla Lys Gly Ala 1155 1160 1165 Ser Gln Ala Asn Asn Ser Ala Gly Tyr ValAsp Ala Asp Gly Asn Lys 1170 1175 1180 Val Ile Tyr Asp Ser Thr Asp AsnLys Tyr Tyr Gln Ala Lys Asn Asp 1185 1190 1195 1200 Gly Thr Val Asp LysThr Lys Glu Val Ala Lys Asp Lys Leu Val Ala 1205 1210 1215 Gln Ala GlnThr Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser 1220 1225 1230 ValIle Asn Lys Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile 1235 12401245 Asn Glu Asp Asn Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp1250 1255 1260 Asn Lys Thr Lys Asn Ala Ala Val Thr Val Gly Asp Leu AsnAla Val 1265 1270 1275 1280 Ala Gln Thr Pro Leu Thr Phe Ala Gly Asp ThrGly Thr Thr Ala Lys 1285 1290 1295 Lys Leu Gly Glu Thr Leu Thr Ile LysGly Gly Gln Thr Asp Thr Asn 1300 1305 1310 Lys Leu Thr Asp Asn Asn IleGly Val Val Ala Gly Thr Asp Gly Phe 1315 1320 1325 Thr Val Lys Leu AlaLys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala 1330 1335 1340 Gly Gly ThrLys Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser 1345 1350 1355 1360Gly Gln Ala Lys Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp 13651370 1375 Leu Gly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys AspThr 1380 1385 1390 Asp Ala Ala Asn Val Gln Gln Leu Asn Glu Val Arg AsnLeu Leu Gly 1395 1400 1405 Leu Gly Asn Ala Gly Asn Asp Asn Ala Asp GlyAsn Gln Val Asn Ile 1410 1415 1420 Ala Asp Ile Lys Lys Asp Pro Asn SerGly Ser Ser Ser Asn Arg Thr 1425 1430 1435 1440 Val Ile Lys Ala Gly ThrVal Leu Gly Gly Lys Gly Asn Asn Asp Thr 1445 1450 1455 Glu Lys Leu AlaThr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly 1460 1465 1470 Asn AlaAsn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys Asp 1475 1480 1485Gly Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr 14901495 1500 Asn Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile AsnGlu 1505 1510 1515 1520 Gln Gly Ile Arg Phe Phe His Val Asn Asp Gly AsnGln Glu Pro Val 1525 1530 1535 Val Gln Gly Arg Asn Gly Ile Asp Ser SerAla Ser Gly Lys His Ser 1540 1545 1550 Val Ala Ile Gly Phe Gln Ala LysAla Asp Gly Glu Ala Ala Val Ala 1555 1560 1565 Ile Gly Arg Gln Thr GlnAla Gly Asn Gln Ser Ile Ala Ile Gly Asp 1570 1575 1580 Asn Ala Gln AlaThr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn 1585 1590 1595 1600 ValVal Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val 1605 16101615 Lys Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr Asp1620 1625 1630 Ala Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile ThrVal Thr 1635 1640 1645 Glu Ser Asn Ser Val Ala Leu Gly Ser Asn Ser AlaIle Ser Ala Gly 1650 1655 1660 Thr His Ala Gly Thr Gln Ala Lys Lys SerAsp Gly Thr Ala Gly Thr 1665 1670 1675 1680 Thr Thr Thr Ala Gly Ala ThrGly Thr Val Lys Gly Phe Ala Gly Gln 1685 1690 1695 Thr Ala Val Gly AlaVal Ser Val Gly Ala Ser Gly Ala Glu Arg Arg 1700 1705 1710 Ile Gln AsnVal Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp Ala 1715 1720 1725 ValAsn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala 1730 17351740 Thr Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala Asn1745 1750 1755 1760 Ala Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met ProGln Ala Tyr 1765 1770 1775 Ile Pro Gly Arg Ser Met Val Thr Gly Gly IleAla Thr His Asn Gly 1780 1785 1790 Gln Gly Ala Val Ala Val Gly Leu SerLys Leu Ser Asp Asn Gly Gln 1795 1800 1805 Trp Val Phe Lys Ile Asn GlySer Ala Asp Thr Gln Gly His Val Gly 1810 1815 1820 Ala Ala Val Gly AlaGly Phe His Phe 1825 1830 5 20 PRT Moraxella catarrhalis UNSURE (17) 5Asn Val Lys Ser Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys 1 5 1015 Xaa Gln Gly Ile 20 6 16 PRT Moraxella catarrhalis 6 Asn Val Lys SerVal Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys 1 5 10 15 7 60 DNAMoraxella catarrhalis 7 aatgtcaaat cagtcattaa caaagaacaa gtaaatgatgccaataaaaa gcaaggcatc 60 8 20 PRT Moraxella catarrhalis 8 Asn Val LysSer Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys 1 5 10 15 Lys GlnGly Ile 20 9 30 PRT Moraxella catarrhalis 9 Met Ile Gly Ala Thr Leu SerGly Ser Ala Tyr Ala Gln Lys Lys Asp 1 5 10 15 Thr Lys His Ile Ala IleGly Glu Gln Asn Gln Pro Arg Arg 20 25 30 10 30 PRT Moraxella catarrhalis10 Ser Gly Thr Ala Lys Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu 1 510 15 Asn Ala Asn Ala Gln Gly Gly Gln Ala Ile Ala Ile Gly Ser 20 25 30

What we claim is:
 1. An isolated and purified outer membrane protein ofa Moraxella strain having an apparent molecular mass of about 200 kDa,as determined by SDS-PAGE or a fragment or an analog thereof.
 2. Theprotein of claim 1 wherein the Moraxella strain is Moraxellacatarrhalis.
 3. The protein of claim 2 wherein the strain is Moraxellacatarrhalis
 4223. 4. The protein of claim 1 containing the amino acidsequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10) for Moraxella catarrhalis strain 4223 or containing thecorresponding amino acid sequence from other Moraxella strains.
 5. Theprotein of claim 1 which is at least about 70 wt % pure.
 6. The proteinof claim 5 which is at least about 95 wt % pure.
 7. The protein of claim1 in the form of an aqueous solution thereof.
 8. The protein of claim 1recognizable by an antibody preparation specific for a peptide havingthe amino acid sequence ofNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10).
 9. The protein of claim 1 having substantially theamino acid composition as shown in Table III.
 10. A purified andisolated nucleic acid molecule encoding an outer membrane protein of astrain of Moraxella having a molecular mass of about 200 kDa, asdetermined by SDS-PAGE, or a fragment or an analog of the outer membraneprotein.
 11. The nucleic acid molecule of claim 10, wherein the strainof Moraxella is a strain of Moraxella catarrhalis.
 12. The nucleic acidmolecule of claim 11, wherein the strain is Moraxella catarrhalis 4223.13. The nucleic acid molecule of claim 10, wherein the encoded proteincontains the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10) for Moraxella catarrhalis strain 4223 or contains thecorresponding amino acid sequence from other Moraxella strains.
 14. Apurified and isolated nucleic acid molecule having a sequence selectedfrom the group consisting of: (a) a DNA sequence as set out in FIG. 6(SEQ ID Nos: 1 and 2), or the complementary sequence thereto; (b) a DNAsequence encoding a 200 kDa protein of a strain of Moraxella andcontaining the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10), or the complementary sequence thereto; (c) a DNAsequence encoding a deduced amino acid sequence as set out in FIG. 6(SEQ ID No: 3), or the complimentary sequence thereto; and (d) anucleotide sequence which hybridizes under stringent conditions to anyone of the sequences defined in (a), (b) or (c).
 15. The nucleic acidmolecule of claim 14, wherein the nucleotide sequence defined in (d) hasat least about 90% sequence identity with any one of the sequencesdefined in (a), (b) or (c).
 16. A vector adapted for transformation of ahost comprising the nucleic acid molecule of claim 10 or
 14. 17. Anexpression vector adapted for transformation of a host comprising thenucleic acid molecule of claim 10 or 14 and expression means operativelycoupled to the nucleic acid molecule for expression by the host of saidouter membrane protein of a strain of Moraxella or the fragment or theanalog of the outer membrane protein.
 18. The expression vector of claim17, wherein the expression means includes a nucleic acid portionencoding a leader sequence for secretion from the host of the outermembrane protein or the fragment or the analog of the outer membraneprotein.
 19. The expression vector of claim 17, wherein the expressionmeans includes a nucleic acid portion encoding a lipidation signal forexpression from the host of a lipidated form of the outer membraneprotein or the fragment or the analog of the outer membrane protein. 20.A transformed host containing an expression vector as claimed in claim17.
 21. A recombinant outer membrane protein or fragment or analogthereof producible by the transformed host of claim
 20. 22. A livevector for delivery of an outer membrane protein of a strain ofMoraxella having a molecular weight of about 200 kDa or a fragment oranalog thereof to a host, comprising a vector containing the nucleicacid molecule of claim 10 or
 14. 23. The live vector of claim 21,wherein the vector is selected from the group consisting of E. coli,Salmonella, Mycobacteria, adenovirus, poxvirus, vaccinia and poliovirus.24. A peptide having no less than six amino acids and no more than 150amino acids and containing an amino acid sequence corresponding to aportion only of an outer membrane protein of a strain of Moraxellahaving a molecular mass of about 200 kDa, as determined by SDS-PAGE, ora fragment or of an analog of the outer membrane protein..
 25. Thepeptide of claim 24, wherein the Moraxella strain is a Moraxellacatarrhalis strain.
 26. The peptide of claim 25, wherein the strain isMoraxella catarrhalis
 4223. 27. The peptide of claim 24 having the aminoacid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile(SEQ ID No: 5) orNH2-Asn-Val-Lys-Ser-Val-lle-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQID No: 6) for the Moraxella catarrhalis 4223 strain or the amino acidsequence for the corresponding peptide for other strains of Moraxella.28. An immunogenic composition, comprising at least one active componentselected from the group consisting of: (A) an isolated and purifiedouter membrane protein of a Moraxella strain having a molecular mass ofabout 200 kDa, as determined by SDS-PAGE, or a fragment or an analogthereof; (B) a purified and isolated nucleic acid molecule encoding anouter membrane protein of a strain of Moraxella having a molecular massof about 200 kDa, as determined by SDS-PAGE or a fragment or an analogthereof; (C) a purified and isolated nucleic acid molecule having asequence selected from the group consisting of: (a) a DNA sequence setout in FIG. 6 (SEQ ID No: 1 or 2), or the complementary sequencethereto; (b) a DNA sequence encoding an about 200 kDa protein of astrain of Moraxella and containing the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10), or the complementary sequence thereto; (c) a DNAsequence encoding an amino acid sequence as set forth in FIG. 6 (SEQ IDNo: 3), or the complimentary sequence thereto; and (d) a nucleotidesequence which hybridizes under stringent conditions to any one of thesequences defined in (a), (b) or (c); (D) a recombinant outer membraneprotein or fragment or analog thereof producible in a transformed hostcontaining an expression vector comprising a nucleic acid molecule asdefined in (B) or (C) and expression means operatively coupled to thenucleic acid molecule for expression by the host of the recombinantouter membrane protein or fragment or analog thereof; (E) a live vector,comprising a vector containing a purified and isolated nucleic acidmolecule encoding a protein of a strain of Moraxella having a molecularmass of about 200 kDa, as determined by SDS-PAGE, or a fragment oranalog thereof; (F) a live vector, comprising a vector containing apurified and isolated nucleic acid molecule having a sequence selectedfrom the group consisting of: (a) a DNA sequence as set out in FIG. 6(SEQ ID No: 1), or the complementary sequence thereto; (b) a DNAsequence encoding an about 200 kDa protein of a strain of Moraxella andcontaining the amino acid sequenceNH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile(SEQ ID No: 10), or the complementary sequence thereto; (c) a DNAsequence encoding an amino acid sequence as set forth in FIG. 6 (SEQ IDNo: 3), or the complimentary sequence thereto; and (d) a nucleotidesequence which hybridizes under stringent conditions to any one of thesequences defined in (a), (b) or (c); and (G) a peptide having no lessthan six amino acids and no more than 150 amino acids and containing anamino acid sequence corresponding to a portion only of an outer membraneprotein of a strain of Moraxella having a molecular mass of about 200kDa, as determined by SDS-PAGE, or of an analog of the outer membraneprotein; and a pharmaceutically acceptable carrier therefor, said atleast one active component producing an immune response whenadministered to a host.
 29. The immunogenic composition of claim 28formulated as a vaccine for in vivo administration to a host to conferprotection against disease caused by a strain of Moraxella.
 30. Theimmunogenic composition of claim 29 wherein the strain is Moraxellacatarrhalis.
 31. The immunogenic composition of claim 29 formulated as amicroparticle, capsule, ISCOM, or liposome preparation.
 32. Theimmunogenic composition of claim 29 in combination with a targetingmolecule for delivery to specific cells of the immune system or tomucosal surfaces.
 33. The immunogenic composition of claim 29 furthercomprising at least one other immunogenic or immunostimulating material.34. The immunogenic composition of claim 33 wherein the at least oneother immunostimulating material is at least one adjuvant.
 35. Theimmunogenic composition of claim 34 wherein the at least one adjuvant isselected from the group consisting of aluminum phosphate, aluminumhydroxide, QS21, Quil A, derivatives and components thereof, ISCOMmatrix, calcium phosphate, calcium hydroxide, zinc hydroxide, aglycolipid analog, an octodecyl ester of an amino acid, a muramyldipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein.36. The immunogenic composition of claim 35 wherein the host is aprimate.
 37. The immunogenic composition of claim 36 wherein the primateis a human.